A dezocine hapten and artificial antigen and preparation method and application thereof

Abstract

The invention discloses a dezocine hapten and an artificial antigen and a preparation method and application thereof. The dezocine artificial hapten and the artificial antigen are synthesized by taking dezocine as a raw material. The obtained antigen has good immunogenicity and reactivity, and the corresponding antibody is easy to obtain when performing animal immunization, and the antigen can be used to prepare a dezocine colloidal gold detection test paper. The dezocine colloidal gold detection test paper provided by the invention can quickly and conveniently detect dezocine in body fluids, has the advantages of low cost, convenience and speed compared with high performance liquid chromatography and the like, can effectively monitor the dosage of dezocine, and reduce the harm caused by the abuse of dezocine, and the detection result is easy to interpret and is suitable for most scenarios and users.

Description

Technical Field

[0001] The invention belongs to the field of rapid immunological detection, and in particular relates to a dezocine hapten and an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Dezocine is a common opioid analgesic in China. It is a κ receptor agonist and a μ receptor antagonist. It mainly stimulates κ receptors to produce analgesic and sedative effects. At present, dezocine has been widely used in postoperative analgesia. It works well with other anesthetics and analgesics and can effectively reduce the pain caused by surgery. In addition to postoperative analgesia, it can also effectively improve the anesthetic effect during surgery by combining it with local anesthetics. Studies have shown that dezocine, as a clinical analgesic, has a significant clinical analgesic effect, which is no less than the same dose of fentanyl or morphine, and the injection of dezocine has fewer adverse reactions, which allows it to be quickly promoted and used.

[0003] Most studies and reports related to dezocine indicate that dezocine is less addictive. Although its addictiveness is not as strong as some common opioid drugs, there are also some cases of dezocine addiction. In these cases, patients are mostly addicted to dezocine due to repeated use, which makes them dependent on dezocine injections. Studies have shown that dezocine has the same action site as some antidepressants and can effectively relieve patients' depression. Most addicted patients say that dezocine can effectively relieve pain, relax the body and mind after injection, and help sleep. These factors promote patients' addiction to dezocine. In addition, dezocine is not strictly controlled in China, making it easy for most patients to obtain it on their own, which greatly increases the chances of long-term use and addiction in patients with certain mental illnesses.

[0004] As for the addiction risk of dezocine, on the one hand, it is necessary to strengthen the control of dezocine drugs, and on the other hand, strengthen patients' self-protection awareness and correct medication habits. In the control of dezocine drugs, convenient and accurate detection methods are the key part of the control.

[0005] After decades of development, immunochromatographic colloidal gold technology has been widely used in the medical and production fields, and has become increasingly mature in the field of drug abuse. There are colloidal gold test strips for most drugs and regulated drugs on the market. Compared with other detection methods, the detection operation of colloidal gold is simpler, the detection cost is lower, and it has a relatively large advantage. Dezocine is widely used in postoperative analgesia and local anesthesia as an opioid analgesic, and the lax supervision of dezocine in China makes dezocine have certain abuse risks. At present, there are no patents and documents for detecting dezocine abuse-related colloidal gold test strips. There is still a certain gap in the use of immunochromatographic colloidal gold technology to monitor dezocine abuse. The use of high-performance liquid chromatography and other technologies to monitor dezocine abuse is complicated and costly. At present, there is no colloidal gold test strip for detecting dezocine and antigens. The present invention mainly innovates and develops a colloidal gold test strip for detecting dezocine and its antigens and haptens, which provides a good detection option for detecting and controlling the abuse of dezocine, helping dezocine addicts get rid of dependence and protecting the physical and mental health of dezocine users. Summary of the invention

[0006] The first object of the present invention is to provide a dezocine hapten in view of the deficiencies in the prior art.

[0007] A dezocine hapten, the structural formula of which is shown in formula (1):

[0008]

[0009] The second object of the present invention is to provide a method for preparing a dezocine hapten, which uses dezocine as a raw material, is protected and deprotected by the Boc group, and then reacts with trifluoroacetic anhydride for esterification, thereby protecting the amino group of the dezocine structure and obtaining a hapten with a carboxyl structure; the present invention adopts the following technical scheme:

[0010] Step (1), dissolving dezocine in tetrahydrofuran, adding triethylamine and di-tert-butyl dicarbonate, and reacting for 3 to 5 hours to obtain product A;

[0011] Step (2), dissolving product A in dichloromethane, adding triethylamine, succinic anhydride, and 4-dimethylaminopyridine (DMAP), reacting at room temperature for 14 to 16 hours, and obtaining an oily substance B after separation by thin layer chromatography;

[0012] Step (3), dissolving the oily substance B in a mixed solution of trifluoroacetic acid and dichloromethane, stirring and reacting for 4 to 6 hours, and then evaporating to dryness to obtain an oily residue C;

[0013] Step (4), dissolving the oily residue C in benzene, adding trifluoroacetic anhydride to react at room temperature for 1 to 2 hours, then reflux in an oil bath for a period of time, and separating by thin layer chromatography to obtain the dezocine artificial hapten.

[0014] Preferably, in step (1), the molar ratio of dezocine, triethylamine and di-tert-butyl dicarbonate is (0.5-2):(1.0-3.5):(0.5-2.5), more preferably 1:2:1.1.

[0015] Preferably, the molar ratio of product A, triethylamine and succinic anhydride in step (2) is (0.5-1.5):(1-4):(1-3), more preferably 1:3:2.

[0016] Preferably, in step (3), the molar volume ratio of the oil B, trifluoroacetic acid and dichloromethane is 0.141 mmol:1 mL:1 mL.

[0017] Preferably, in step (4), the molar ratio of the oily residue C to trifluoroacetic anhydride is (0.8-1.3):(1.7-2.4), more preferably 1:2.

[0018] Preferably, the reflux reaction time in step (4) is 3 h.

[0019] The third object of the present invention is to provide a dezocine artificial antigen, which is obtained by coupling the above-mentioned dezocine hapten with bovine serum albumin and then purifying it, and its structural formula is shown in formula (2):

[0020]

[0021] The fourth object of the present invention is to provide a method for preparing a dezocine artificial antigen, using the following technical scheme:

[0022] The dezocine artificial hapten is mixed with N-hydroxysuccinimide (NHS) and EDCI and allowed to react at room temperature for 20 to 24 hours, then added into a bovine serum albumin (BSA) solution and stirred for reaction for 3 to 5 hours, and dialyzed in a sodium carbonate solution with a pH value of 10.00 to 13.00 and a mass percentage concentration of 0.3 to 0.7% and a PBS with a concentration of 0.01 to 0.02 M in sequence, and the dezocine artificial antigen is obtained after centrifugal precipitation.

[0023] Preferably, the molar ratio of dezocine artificial hapten, N-hydroxysuccinimide and carbodiimide is (0.8-1.3):(1.2-1.8):(1.7-2.1), more preferably 1:1.5:2.

[0024] Preferably, the concentration of bovine serum albumin is 4 to 7 mg / ml, more preferably 5 mg / ml.

[0025] The fifth object of the present invention is to provide a dezocine monoclonal antibody obtained by immunizing animals with dezocine artificial antigens.

[0026] Preferably, the preparation process of the dezocine monoclonal antibody of the present invention is as follows: the prepared dezocine artificial antigen is purified and diluted, and then mixed with an adjuvant that enhances immunogenicity and injected into the subcutaneous tissue of the back and thigh muscle of healthy rabbits. The injection is repeated every 3 to 4 weeks, and fresh rabbit blood is collected 7 to 9 days after each injection. After separating the rabbit blood, the binding titer of the antibody obtained in the serum is detected by ELISA. After obtaining the antibody serum with an ideal binding titer, the serum is purified to obtain the dezocine polyclonal antibody, and then the mouse immune-activated B cell fusion tumor cells are used for screening by monoclonal technology, and the mouse ascites is separated and purified to obtain the dezocine monoclonal antibody.

[0027] The sixth object of the present invention is to provide a dezocine colloidal gold test paper, comprising a sample pad, a gold label pad, a test line and a quality control line; the test line is obtained by spotting the dezocine antigen as a raw material.

[0028] Preferably, the preparation process of the gold labeling raw material used for the gold label pad in the dezocine test paper is as follows: a colloidal gold solution adjusted to a pH value of 9.0 is added to a dezocine monoclonal antibody solution with an appropriate labeling amount, and a buffer solution is added after mixing, and the mixture is centrifuged at 12000r / min for 30min, and the precipitate is removed and resuspended with a buffer solution, and the centrifugation and resuspension operation is repeated 3 times to obtain a stable colloidal gold labeled protein, and the labeled protein is purified by filtration and then eluted to obtain a colloidal gold labeled raw material solution used for the gold labeling.

[0029] Preferably, the quality control line raw materials of the dezocine colloidal gold test paper of the present invention are goat anti-rabbit IgG and goat anti-mouse IgG, and the concentrations of the quality control line spray solution after dilution with phosphate buffer solution are 0.5-2.0 mg / ml and 0.1-0.5 mg / ml respectively, and the mixed solution is evenly sprayed on the NC membrane to obtain the quality control line.

[0030] Preferably, the detection line of the dezocine colloidal gold test paper of the present invention is obtained by uniformly spraying 0.1-2.0 mg / ml of dezocine antigen as a spraying raw material on the NC membrane.

[0031] The dezocine colloidal gold test paper of the present invention can perform qualitative determination of dezocine and its antigen, has high sensitivity, and the higher the concentration of dezocine, the lower the intensity of the detection line, and its detection limit is 1000ng / mL. The interpretation rules of the dezocine test paper in the present invention are as follows:

[0032] Valid interpretation: When the quality control line, i.e., line C, shows a purple-red line and the color of the line is obvious, it proves that the interpretation of the test paper is valid;

[0033] Negative: When the quality control line, i.e., C line, and the test line, i.e., T line, appear purple-red and the line colors are obvious, it proves that there is no dezocine and its antigen in the test sample, and the reading is negative;

[0034] Positive: When the quality control line, i.e., C line, appears purple-red and the line color is obvious, and the test line, i.e., T line, does not show purple-red lines, it proves that dezocine and its antigen exist in the test sample, and it is judged as positive.

[0035] The present invention has the following beneficial effects:

[0036] The invention uses dezocine as a raw material to synthesize dezocine artificial hapten and artificial antigen. The obtained antigen has good immunogenicity and reactivity, and the corresponding antibody is easy to obtain when performing animal immunization, and can be used to prepare dezocine colloidal gold detection test paper. The dezocine colloidal gold detection test paper provided by the invention can quickly and easily detect dezocine in body fluids, and has the advantages of low cost, convenience and speed compared with high performance liquid chromatography and the like, can effectively monitor the dosage of dezocine, reduce the harm caused by the abuse of dezocine, and the detection result is easy to interpret and is suitable for most scenarios and users. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a liquid chromatogram of the dezocine artificial hapten I obtained in Example 1, wherein mAU represents milliabsorptivity unit, and min represents the time unit minute.

[0038] Figure 2 This is the ESI-MS spectrum of the dezocine artificial hapten I obtained in Example 1, wherein Intens. represents signal intensity and m / z represents mass-to-charge ratio. DETAILED DESCRIPTION

[0039] The present invention is further analyzed below in conjunction with specific embodiments.

[0040] Example 1: Preparation of Dezocine Artificial Hapten

[0041] 1. Weigh 200 mg (0.816 mmol) of dezocine and place it in a 50 ml single-mouth round-bottom flask. Add 3 ml of tetrahydrofuran to dissolve it. At this time, it is a colorless and transparent solution. After cooling the reaction solution in an ice-water bath, add 227 μL (1.633 mmol) of triethylamine, then dissolve 206 μL (0.898 mmol) of di-tert-butyl dicarbonate in 2 ml of tetrahydrofuran to obtain a solution, add it dropwise to the reaction solution, slowly heat it to 25°C, and continue stirring the reaction for 4 hours. After the reaction is completed, evaporate the reaction solution under reduced pressure to dryness to obtain a colorless and transparent solution of 298 mg, and directly proceed to the next step of reaction.

[0042] 2. Dissolve 298 mg (calculated according to theoretical value) (0.816 mmol) of the product in the previous step in 15 ml of dichloromethane, add 341 μL (2.448 mmol) of triethylamine, 163 mg (1.630 mmol) of succinic anhydride and 30 mg of DMAP, stir at room temperature for 16 hours, evaporate the solvent under reduced pressure after the reaction is completed, add 15 ml of purified water and 15 ml of dichloromethane for extraction, collect the organic phase, extract the aqueous phase with 15 ml*2 of dichloromethane, combine the organic phases, wash with 30 ml of purified water and 30 ml of saturated brine, collect the organic phases, dry and filter, evaporate to dryness under reduced pressure to obtain a light yellow oil, and separate another light yellow oil 188 mg (0.423 mmol) by thin layer chromatography (solvent and eluent are anhydrous ethanol, developing solvent is dichloromethane: 95% ethanol: 1,4-dioxane: ammonia water = 10:8:1:1, product Rf = 0.4).

[0043] 3. Dissolve 188 mg (0.423 mmol) of the light yellow oil in 3 ml of trifluoroacetic acid and 3 ml of dichloromethane, stir at room temperature for 5 hours, evaporate to dryness under reduced pressure to obtain 132 mg of a yellow oily residue, which is directly used for the next step.

[0044] 4. Dissolve 152 mg (calculated according to theoretical value) (0.423 mmol) of the yellow oily residue in the previous step in 5 ml of benzene, place in an ice-water bath with stirring, and then slowly add 118 μL (0.846 mmol) of trifluoroacetic anhydride, and white smoke is generated. Then, stir at room temperature for reaction for 1 hour, and then stir and reflux in an oil bath at 80°C for reaction for 3 hours. End the reaction, evaporate the solvent under reduced pressure, and separate 143 mg (0.324 mmol) of a yellow oily substance, i.e., dezocine artificial hapten, by thin layer chromatography (solvent and eluent are anhydrous ethanol, developing solvent is dichloromethane: 95% ethanol: 1,4-dioxane: ammonia water = 10:8:1:1, product Rf = 0.3).

[0045] like Figure 1 As shown, the purity of the purified dezocine artificial hapten I is above 99%, which meets the purity requirement and can be used for the next step reaction.

[0046] like Figure 2 As shown, the mass-to-charge ratio (m / z) of the molecular ion peak of the dezocine artificial hapten I obtained in Example 1 is 441.44, which is consistent with its theoretical molecular weight. It can be preliminarily determined that the structural formula of the dezocine artificial hapten is Formula I.

[0047] Example 2: Preparation of Dezocine Artificial Antigen

[0048] 1. Dissolve 143 mg (0.324 mmol) of the above-mentioned dezocine artificial hapten in 7.15 ml of tetrahydrofuran, add 56 mg (0.486 mmol) of NHS and 124 mg (0.648 mmol) of EDCI, stir and react at room temperature in the dark for 24 hours, centrifuge at 10000 rpm for 15 min, discard the precipitate, take the supernatant and dry it, dissolve the residue in 2.86 ml of dimethyl sulfoxide to obtain an activated dezocine hapten solution.

[0049] 2. Weigh 175 mg BSA and dissolve it in 35 ml 0.01 M PBS buffer to prepare a 5 mg / ml BSA PBS solution.

[0050] 3. Slowly add the activated dezocine hapten solution to the BSA solution prepared in the previous step. Stir the BSA solution rapidly during the addition process. Then seal the container and avoid light, stir and react at room temperature for 4 hours.

[0051] 4. The reaction solution was transferred into a dialysis bag, and dialyzed in a 0.5% sodium carbonate solution with a pH of 12.00 for two days, and then transferred into 0.01M PBS for 3 days, twice a day. The dialysate was centrifuged to remove the precipitate to obtain the dezocine artificial antigen, which was stored at -20°C for later use.

[0052] Example 3: Preparation of Dezocine Monoclonal Antibody

[0053] The prepared dezocine artificial antigen was purified, diluted to 2 mg / ml with phosphate buffer solution, and mixed with Freund's adjuvant in a ratio of 1:1. 0.3 mg of the artificial antigen mixture was injected into the back subcutaneous tissue and thigh muscle of healthy rabbits every 3 to 4 weeks. Fresh rabbit blood was collected 7 to 9 days after each injection. The rabbit blood was separated by centrifugation at 12000 rpm for 15 minutes in a high-speed centrifuge. The binding titer of the antibody obtained in the serum was detected by ELISA. When the antibody titer reached 1:200 or above, the serum was purified to obtain dezocine polyclonal antibodies. Then, mouse immune-activated B cell fusion tumor cells were used for screening by monoclonal technology, and the mouse ascites was separated and purified to obtain dezocine monoclonal antibodies.

[0054] Example 4: Preparation of Dezocine Colloidal Gold Test Paper

[0055] 1. The prepared dezocine artificial antigen is diluted with phosphate buffer solution to a concentration of 0.1-2 mg / ml, sucrose is added to play a role in biological protection, and the mixed solution is evenly sprayed on the NC membrane at a spray volume of 1.0-1.5 ul / cm to obtain the test line; the goat anti-rabbit IgG and goat anti-mouse IgG in the quality control line raw materials are 0.5-2.0 mg / ml and 0.1-0.5 mg / ml respectively, and after adding sucrose, the solution is evenly sprayed on the NC membrane at a spray volume of 1.0-1.5 ul / cm, and the coated NC membrane is obtained after being placed in an oven at about 45°C for 16-22 hours.

[0056] 2. Mix the colloidal gold solution with adjusted pH value with the dezocine monoclonal antibody solution, react for 15 to 20 minutes, add BSA solution for blocking and react for another 15 to 20 minutes, centrifuge at 10,000 rpm for 30 minutes, and dilute the precipitate with buffer to obtain the labeled dezocine monoclonal antibody raw material.

[0057] 3. Add the labeled dezocine monoclonal antibody raw material, rabbit IgG and mouse IgG into diluent, trehalose and sucrose, dilute to a suitable concentration, and then spray evenly on the polyester film at a spray volume of 1.0-1.5ul / cm. Place in an oven at about 37°C for 12-22 hours to obtain the gold label pad after spraying.

[0058] 4. Place the filter paper, NC membrane, gold label pad and sample pad on the PVC sheet cardboard from top to bottom, with the filter paper and gold label pad placed on the NC membrane, and the sample pad placed on the gold label pad.

[0059] Detection principle: Dezocine, as a commonly used clinical analgesic, is often injected into the patient's body. About 80% of it will be excreted in the form of urine, of which 1% is the prototype and the rest is a conjugate of glucuronide. The colloidal gold product of the present invention mainly detects dezocine and its metabolites in urine, and uses a competitive method to detect dezocine and its hapten in urine or other body fluids. If the urine to be tested contains dezocine, the dezocine in the urine will first bind to the labeled dezocine monoclonal antibody, and the labeled dezocine monoclonal antibody has already bound to the dezocine in the urine, so it does not bind to the dezocine antigen coated with the detection line, then the detection line is wireless, and the labeled rabbit IgG and mouse IgG are combined with the goat anti-rabbit IgG and goat anti-mouse IgG of the quality control line, and the quality control line has wires. If the urine contains dezocine and its metabolites, the C line has wires and the T line has no wires; if the urine does not contain dezocine and its metabolites, both the C line and the T line have wires.

[0060] How to use the product: The product should be kept in a dry and cool place. Use a dropper to drop three drops of the sample solution to be tested vertically onto the sample pad and wait for 5 minutes before reading. If the C line has a wire and the T line has a wire, it is negative; if the C line has a wire and the T line has no wire, it is positive.

[0061] Test example: Dezocine colloidal gold test paper performance test

[0062] 1. Sensitivity: Dezocine solutions of different concentrations (0 ng / ml, 500 ng / ml, 1000 ng / ml, 1500 ng / ml) were prepared to test the performance of the dezocine colloidal gold test paper product prepared by the present invention. In the test results, positive was indicated by "+", and negative was indicated by "-". Each concentration of dezocine solution was tested 3 times. The results of dezocine colloidal gold test paper testing dezocine solutions of different concentrations were as follows:

[0063] Table 1 Results of dezocine colloidal gold test strips for detecting dezocine solutions of different concentrations

[0064] Dezocine concentration (ng / ml) Test result (+ or -) 0 -，-，- 500 -，-，- 1000 +，+，+ 1500 +，+，+

[0065] 2. Stability: The prepared dezocine colloidal gold test paper was stored at -2°C and 37°C for 3 days, and the dezocine colloidal gold test paper stored at room temperature for 3 days was used as a control. Dezocine solutions with concentrations of 0ng / ml, 500ng / ml, and 1500ng / ml were tested respectively. The test results were graded into ten intensities from G1 to G10. The test was repeated three times and the average value was taken. The test line strength results of the dezocine colloidal gold test paper stored under three temperature conditions are as follows:

[0066] Table 2 Test line strength results of dezocine colloidal gold test paper stored at different temperatures

[0067]

[0068] 3. Specificity: 10ug / ml standard solutions of common drugs such as morphine, ketamine, methamphetamine and other drugs were used as test solutions and tested using the product of the present invention. The test results were represented by "+" for positive and "-" for negative. The test was repeated three times. The test results of dezocine colloidal gold test paper are as follows:

[0069] Table 3 Detection results of dezocine colloidal gold test paper on common drugs

[0070] Cross-substance (10ug / ml) Test result (+ or -) morphine -，-，- Ketamine -，-，- Methamphetamine -，-，- Ephedrine -，-，- Pseudoephedrine -，-，- Diazepam -，-，- Phenobarbital -，-，- Tramadol -，-，- Benzoylecgonine -，-，- Procaine -，-，- Methadone -，-，- Gatifloxacin -，-，- Ranitidine -，-，-

[0071] 4. Detection rate: 200 clinical urine samples were collected and tested with the dezocine colloidal gold test paper prepared by the present invention and LC / MS, respectively. The number of positive samples detected by the test paper was divided by the number of positive samples measured by LC / MS to obtain the positive detection rate of the test paper product. The results and detection rate of clinical urine samples tested by dezocine colloidal gold test paper are as follows:

[0072] Table 4 Test results of dezocine colloidal gold test strips on clinical urine samples

[0073] Detection Methods Number of positive results Number of negative results Dezocine colloidal gold test paper 39 161 LC / MS 42 158

[0074] The positive detection rate of the dezocine colloidal gold test paper is: 39 / 42*100%=92.85%

[0075] From the performance test of the dezocine colloidal gold test paper, it can be concluded that the minimum detection limit of the test paper prepared by the present invention can reach 1000ng / ml, with high sensitivity, and no overlap with the test results of other common drugs and medicines, and good specificity. The positive detection rate for clinical urine samples is as high as 92.85%, and the dezocine in the urine of patients with dezocine abuse can be detected in time, thereby discovering the abuse of dezocine, which plays a key role in drug supervision and abuse prevention.

Claims

1. A dezocine hapten, characterized in that: Its structural formula is shown in formula (1):

2. The method for preparing the dezocine hapten according to claim 1, characterized in that: The preparation method comprises the following steps: Step (1), dissolving dezocine in tetrahydrofuran, adding triethylamine and di-tert-butyl dicarbonate, and reacting for 3 to 5 hours to obtain product A; Step (2), dissolving product A in dichloromethane, adding triethylamine, succinic anhydride, and 4-dimethylaminopyridine DMAP, reacting at room temperature for 14 to 16 hours, and obtaining an oily substance B after separation by thin layer chromatography; Step (3), dissolving the oily substance B in a mixed solution of trifluoroacetic acid and dichloromethane, stirring for reaction for 4 to 6 hours, and then evaporating to dryness to obtain an oily residue C; Step (4), dissolving the oily residue C in benzene, adding trifluoroacetic anhydride to react at room temperature for 1 to 2 hours, then reflux in an oil bath for a period of time, and separating by thin layer chromatography to obtain the dezocine artificial hapten.

3. The preparation method according to claim 2, characterized in that: The molar ratio of dezocine, triethylamine and di-tert-butyl dicarbonate in step (1) is (0.5-2):(1.0-3.5):(0.5-2.5).

4. The preparation method according to claim 2 or 3, characterized in that: In step (2), the molar ratio of product A, triethylamine and succinic anhydride is (0.5-1.5):(1-4):(1-3).

5. The preparation method according to claim 2, characterized in that: The molar volume ratio of the oily substance B, trifluoroacetic acid and dichloromethane in step (3) is 0.141 mmol:1 mL:1 mL; the molar ratio of the oily residue C and trifluoroacetic anhydride in step (4) is (0.8-1.3):(1.7-2.4).

6. A dezocine artificial antigen, characterized in that: The dezocine hapten described in claim 1 is coupled to bovine serum albumin and then purified, and its structural formula is shown in formula (2):

7. The method for preparing the dezocine artificial antigen according to claim 6, characterized in that: The preparation method comprises the following steps: The dezocine artificial hapten, N-hydroxysuccinimide NHS and carbodiimide EDCI are mixed and reacted at room temperature for 20 to 24 hours, then added into a bovine serum albumin BSA solution and stirred for reaction for 3 to 5 hours, and dialyzed in a sodium carbonate solution with a pH value of 10.00 to 13.00 and a mass percentage concentration of 0.3 to 0.7% and a PBS with a concentration of 0.01 to 0.02M in sequence, and the dezocine artificial antigen is obtained after centrifugal precipitation.

8. The preparation method according to claim 7, characterized in that: The molar ratio of dezocine artificial hapten, N-hydroxysuccinimide and carbodiimide is (0.8-1.3): (1.2-1.8): (1.7-2.1).

9. A dezocine colloidal gold test paper, comprising a sample pad, a gold label pad, a test line and a quality control line, characterized in that: The test line is obtained by using the dezocine antigen as the raw material for spot filming according to claim 6.

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