DIMETHYLTRYPTAMINE HAPTEN, ARTIFICIAL ANTIGEN AND PREPARATION METHODS AND APPLICATION THEREOF
20230393159 · 2023-12-07
Assignee
Inventors
- Cheng Liu (Zhejiang, CN)
- Haijian WANG (Zhejiang, CN)
- Jiayi GAO (Zhejiang, CN)
- Fei GAO (Zhejiang, CN)
- Weike LU (Zhejiang, CN)
Cpc classification
G01N33/577
PHYSICS
International classification
G01N33/94
PHYSICS
G01N33/577
PHYSICS
Abstract
The present invention relates to the field of detection of dimethyltryptamine. In order to solve the problems in the prior art that liquid chromatography-tandem mass spectrometry for detection of dimethyltryptamine has high requirements for instruments and operators and is difficult to popularize and low in detection speed, the present invention discloses a dimethyltryptamine hapten, an artificial antigen and preparation methods and application thereof. The dimethyltryptamine hapten is obtained by a reaction of N,N-dimethyl-5-hydroxytryptamine as a precursor and glutaric anhydride. The artificial dimethyltryptamine antigen obtained from the dimethyltryptamine hapten can be used for preparing a dimethyltryptamine monoclonal antibody and a dimethyltryptamine colloidal gold-fluorescence test paper. The test paper has high detection sensitivity, can be used for detecting dimethyltryptamine in urine, blood, saliva and hair rapidly, has low requirements for instruments, and is easy and convenient to operate and conducive to wide popularization of drug detection.
Claims
1. A dimethyltryptamine hapten, wherein the dimethyltryptamine hapten has a molecular structural formula as shown in Formula (I), ##STR00004##
2. A preparation method of the dimethyltryptamine hapten according to claim 1, comprising the following steps: (1) dissolving N,N-dimethyl-5-hydroxytryptamine as a precursor in benzene, adding trifluoroacetic anhydride to carry out a first reaction under heating, cooling a first reaction solution to room temperature after the first reaction under heating is completed, and removing the benzene to obtain a light-yellow oily compound A; and (2) dissolving the light-yellow oily compound A in pyridine, adding glutaric anhydride to further carry out a second reaction under heating, cooling a second reaction solution to room temperature after the second reaction under heating is completed, and removing the pyridine, followed by separation to obtain a yellow oily compound, namely the dimethyltryptamine hapten.
3. The preparation method of the dimethyltryptamine hapten according to claim 2, wherein in the step (1), the N,N-dimethyl-5-hydroxytryptamine is dissolved as a precursor in the benzene to obtain a solution with a concentration of 0.15-0.20 mmol/mL, and the solution is stirred below 0° C.; then the trifluoroacetic anhydride is slowly added, wherein a molar ratio of the N,N-dimethyl-5-hydroxytryptamine to the trifluoroacetic anhydride is 1:(1-1.2); the temperature is slowly raised to room temperature to carry out a reaction under stirring, and then the temperature is raised to carry out the reaction under stirring; and after the reaction under heating is completed, a reaction solution for the reaction under stirring is cooled to room temperature, and an organic solvent is dried under reduced pressure to obtain the light yellow oily compound A.
4. The preparation method of the dimethyltryptamine hapten according to claim 2, wherein in the step (2), the light yellow oily compound A is dissolved in the pyridine; the glutaric anhydride is added to carry out a reaction under heating and reflux stirring; after the reaction under heating and reflux stirring is completed, a reaction solution for the reaction under heating and reflux stirring is cooled to room temperature, and a solvent is dried under reduced pressure; and then separation is performed by thin-layer chromatography to obtain the yellow oily compound, namely the dimethyltryptamine hapten.
5. An artificial dimethyltryptamine antigen, wherein the artificial dimethyltryptamine antigen is obtained by coupling the dimethyltryptamine hapten according to claim 1 to a carrier protein and has a molecular structural formula as shown in Formula (II), ##STR00005## wherein in the Formula (II), protein refers to the carrier protein.
6. The artificial dimethyltryptamine antigen according to claim 5, wherein the carrier protein is one of bovine serum albumin, bovine γ globulin, bovine thyroglobulin, keyhole limpet hemocyanin and ovalbumin.
7. A preparation method of the artificial dimethyltryptamine antigen according to claim 5, comprising the following steps: (1) dissolving the dimethyltryptamine hapten in N,N-dimethylformamide to obtain a solution with a concentration of 0.045-0.050 mmol/mL, sequentially adding N-hydroxysuccinimide and dicyclohexylcarbodiimide to carry out a reaction under stirring at room temperature, and performing centrifugation to obtain a supernatant recorded as a solution A, wherein a molar ratio of the dimethyltryptamine hapten to the dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 1:(1-1.2):(1.1-1.3); (2) dissolving the carrier protein in PBS buffer to obtain a solution B; (3) slowly adding the solution A dropwise into the solution B under stirring, followed by placement and storage overnight to obtain an artificial antigen mixed solution; and (4) placing the artificial antigen mixed solution in a dialysis bag for dialysis in an alkaline dialysis solution for 2-3 times, and then transferring the artificial antigen mixed solution to the PBS buffer for dialysis for 6-7 times, wherein the dialysis is performed for more than 2 h each time; and after the dialysis is completed, performing centrifugation to obtain a supernatant, namely the artificial dimethyltryptamine antigen, wherein the PBS buffer has a concentration of 0.1 mol/L and a pH value of 7.2-7.4, and the alkaline dialysis solution is a sodium carbonate solution with a pH value of 11.95-12.05.
8. Application of the artificial dimethyltryptamine antigen according to claim 5 in preparation of a dimethyltryptamine monoclonal antibody.
9. Application of the artificial dimethyltryptamine antigen according to claim 5 in preparation of a dimethyltryptamine colloidal gold-fluorescence test paper.
10. The application of the artificial dimethyltryptamine antigen in preparation of the dimethyltryptamine colloidal gold-fluorescence test paper according to claim 9, wherein a test line is obtained by dotting the artificial dimethyltryptamine antigen as a raw material, a quality control line is obtained by dotting goat anti-mouse IgG or goat anti-rabbit IgG as a raw material, and a binding pad is obtained by spraying a dimethyltryptamine monoclonal antibody-colloidal gold complex and a dimethyltryptamine monoclonal antibody-fluorescein complex on the binding pad.
11. A preparation method of the artificial dimethyltryptamine antigen according to claim 6, comprising the following steps: (1) dissolving the dimethyltryptamine hapten in N,N-dimethylformamide to obtain a solution with a concentration of 0.045-0.050 mmol/mL, sequentially adding N-hydroxysuccinimide and dicyclohexylcarbodiimide to carry out a reaction under stirring at room temperature, and performing centrifugation to obtain a supernatant recorded as a solution A, wherein a molar ratio of the dimethyltryptamine hapten to the dicyclohexylcarbodiimide to the N-hydroxysuccinimide is 1:(1-1.2):(1.1-1.3); (2) dissolving the carrier protein in PBS buffer to obtain a solution B; (3) slowly adding the solution A dropwise into the solution B under stirring, followed by placement and storage overnight to obtain an artificial antigen mixed solution; and (4) placing the artificial antigen mixed solution in a dialysis bag for dialysis in an alkaline dialysis solution for 2-3 times, and then transferring the artificial antigen mixed solution to the PBS buffer for dialysis for 6-7 times, wherein the dialysis is performed for more than 2 h each time; and after the dialysis is completed, performing centrifugation to obtain a supernatant, namely the artificial dimethyltryptamine antigen, wherein the PBS buffer has a concentration of 0.1 mol/L and a pH value of 7.2-7.4, and the alkaline dialysis solution is a sodium carbonate solution with a pH value of 11.95-12.05.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0041]
[0042]
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0043] The present invention is further described below in combination with specific implementation methods.
[0044] A PBS buffer used in the following embodiments is prepared by dissolving 14.5 g of disodium hydrogen phosphate dodecahydrate, 43.875 g of sodium chloride and 1.495 g of sodium dihydrogen phosphate dihydrate in double distilled water to a constant volume of 5.0 L, and has a pH value of 7.4. An alkaline dialysis solution is prepared in the following process: adjusting the pH value of a sodium carbonate aqueous solution with a mass fraction of 0.5% to 12.00 with a NaOH solution with a concentration of 2 mol/L.
Example 1
[0045] (1) Preparation of a Dimethyltryptamine Hapten: [0046] A. 0.98 mmol of N,N-dimethyl-5-hydroxytryptamine was added as a precursor into a 50 mL one-mouth round-bottom flask, 5 mL of benzene was added for dissolution to obtain a solution, and the solution was stirred in an ice water bath at 0° C. for 5 min. 1.08 mmol of trifluoroacetic anhydride was slowly added, the temperature was slowly raised to room temperature to carry out a reaction under stirring for 1 h, and then the reaction was carried out in an oil bath under stirring at 78° C. for 3 h and monitored by thin-layer chromatography, where ethyl acetate was used as a chromatographic solution, and the Rf value of a product was 0.9. The reaction was terminated when basically completed, and a reaction solution was cooled to room temperature and then dried under a reduced pressure of −0.1 MPa at 50° C. to obtain a light-yellow oily compound A [0047] B. 0.94 mmol of the light-yellow oily compound A obtained in the previous step was placed in a 50 mL single-mouth round-bottomed flask, 10 mL of pyridine was added for dissolution, and 0.94 mmol of glutaric anhydride was added to obtain a mixture. The mixture was placed in an oil bath to carry out a reaction under reflux stirring at 105° C. for 18 h, and the reaction was monitored by thin-layer chromatography, where a mixture of dichloromethane, ammonia, 95% ethanol and 1,4-dioxane at a ratio of 10:1:8:1 was used as a chromatographic solution, and the Rf value of a product was 0.2. The reaction was terminated after basically completed, a reaction solution was cooled to room temperature, a solvent was dried under reduced pressure, and separation was performed by thin-layer chromatography to obtain a yellow oily compound, where anhydrous ethanol was used as a solvent and an eluent, a mixture of dichloromethane, ammonia, 95% ethanol and 1,4-dioxane at a ratio of 10:1:8:1 was used as a chromatographic solution, the Rf value of the product was 0.2, and the yellow oily compound was the dimethyltryptamine hapten.
[0048] The dimethyltryptamine hapten obtained in step (1) was separately analyzed by high performance liquid chromatography (HPLC) and high resolution mass spectrometry (ESI-MS).
[0049] (2) Preparation of an Artificial Dimethyltryptamine Antigen: [0050] A. 0.36 mmol of the dimethyltryptamine hapten prepared in step (1) was placed in a 25 mL single-mouth round-bottomed flask, 7.5 mL of N,N-dimethylformamide (DMF) was added, then 0.43 mmol of dicyclohexylcarbodiimide (DCC) and 0.43 mmol of N-hydroxysuccinimide (NHS) were added to carry out a reaction under stirring at room temperature for 15 h. After the reaction was completed, centrifugation was performed at 8,000 r/min and 4° C. for 15 min, and a supernatant was collected and recorded as a solution A. [0051] B. 0.375 g of bovine serum albumin (BSA) was weighed and dissolved in 75 mL of a PBS solution to obtain a BSA solution recorded as a solution B. [0052] C. The solution A was slowly added dropwise into the solution B at a volume ratio of 1:10 under rapid stirring to obtain a mixed solution, the obtained mixed solution was placed and stored at 4° C. overnight to obtain an immunogen and coating antigen (DMT-BSA) mixed solution, namely an artificial antigen mixed solution, and then whether the hapten was successfully coupled to the BSA was determined by ultraviolet scanning. [0053] D. The successfully coupled artificial antigen mixed solution was transferred into a dialysis bag for dialysis in an alkaline dialysis solution for 24 h under stirring, where the dialysis was repeated for two times. [0054] E. After the dialysis in the alkaline dialysis solution, the artificial antigen mixed solution was subjected to dialysis in a PBS solution for 7 times for 2 h each time, and after the dialysis was completed, centrifugation was performed to obtain a supernatant, namely the artificial dimethyltryptamine antigen.
Example 2
[0055] (1) Preparation of a dimethyltryptamine hapten: The molar ratio of the N,N-dimethyl-5-hydroxytryptamine to the trifluoroacetic anhydride was 1:1, and other preparation conditions were the same as those in Example 1. [0056] (2) Preparation of an artificial dimethyltryptamine antigen: The molar ratio of the dimethyltryptamine hapten to the DCC to the NHS was 1:1:1.1, and other preparation conditions were the same as those in Example 1.
Example 3
[0057] (1) Preparation of a dimethyltryptamine hapten: The molar ratio of the N,N-dimethyl-5-hydroxytryptamine to the trifluoroacetic anhydride was 1:1.2, and other preparation conditions were the same as those in Example 1. [0058] (2) Preparation of an artificial dimethyltryptamine antigen: The molar ratio of the dimethyltryptamine hapten to the DCC to the NHS was 1:1.2:1.3, and other preparation conditions were the same as those in Example 1.
Example 4
[0059] Preparation of a Dimethyltryptamine Monoclonal Antibody:
[0060] The dimethyltryptamine antigen obtained in Example 1 was diluted to 1 mg/mL with a PBS buffer and mixed with an immune adjuvant at a volume ratio of 1:1 to obtain a mixture, and the mixture was subcutaneously injected into mice to repeatedly immunize the mice every 2 weeks. Serum was isolated, the valence of an antibody in the serum was detected by indirect ELISA, and when the valence of the antibody was smaller than 1:128, the serum was collected and purified to obtain a polyclonal antibody. Then myeloma cells of the immunized mice were fused with spleen B cells under the action of polyethylene glycol as a fusion promoter and screened by a monoclonal cell technology to obtain hybridoma cells, the hybridoma cells were intraperitoneally injected and inoculated into the mice, and ascites was collected 2 weeks later to obtain the dimethyltryptamine monoclonal antibody.
Example 5
[0061] Preparation of a dimethyltryptamine monoclonal antibody: The dimethyltryptamine monoclonal antibody was prepared by using the dimethyltryptamine antigen obtained in Example 2, and other preparation processes were the same as those in Example 4.
Example 6
[0062] Preparation of a dimethyltryptamine monoclonal antibody: The dimethyltryptamine monoclonal antibody was prepared by using the dimethyltryptamine antigen obtained in Example 3, and other preparation processes were the same as those in Example 4.
Example 7
[0063] Preparation of a Dimethyltryptamine Colloidal Gold-Fluorescence Test Paper: [0064] A. A test line and a quality control line: 0.2 mg/mL of the dimethyltryptamine antigen obtained in Example 1 was sprayed on a nitrocellulose membrane at a spraying rate of 1.0 μL/cm to serve as the test line, and 1.0 mg/mL of goat anti-rabbit IgG and 0.1 mg/mL of goat anti-mouse IgG were sprayed on the nitrocellulose membrane at a spraying rate of 1.0 μL/cm to serve as the quality control line. [0065] B. A dimethyltryptamine monoclonal antibody-colloidal gold complex: The dimethyltryptamine monoclonal antibody obtained in Example 4 was dissolved in a colloidal gold solution to obtain a dimethyltryptamine monoclonal antibody solution, and the pH value of the colloidal gold solution was adjusted to 7.6. 1 mL of the dimethyltryptamine monoclonal antibody solution was added and stirred, and a 1% PEG2000 buffer with a volume of 10 mL was added to obtain a mixed solution. The mixed solution was subjected to centrifugation at 100,000 g at 4° C. for 60 min, a precipitate was removed, and resuspension was performed with a PEG2000 buffer repeatedly for 3 times to obtain a colloidal gold labeled protein. Then, the colloidal gold labeled protein was purified by a gel filtration method and eluted with a PBS buffer containing 1% of BSA and 0.02% of sodium azide, and the dimethyltryptamine monoclonal antibody-colloidal gold complex was collected. [0066] C. A dimethyltryptamine monoclonal antibody-fluorescein complex: The dimethyltryptamine monoclonal antibody obtained in Example 4 was diluted to 10 mg/mL with 0.025 mol/L of CB with a pH value of 9.0 to obtain an antibody solution, and the antibody solution was placed in a dialysis bag. An opening of the dialysis bag was tightly tied with only a few gaps retained. An Alexa Fluor® dye solution with a concentration of 5 μg/mL was prepared by dissolving an Alexa Fluor® dye in a PBS buffer and then added into a beaker, where the volume of the Alexa Fluor® dye solution was 10 times higher than that of the antibody solution. The dialysis bag was immersed in the Alexa Fluor® dye solution. Binding was performed under slow stirring by an electromagnetic stirrer at 4° C. for 24 h, the dialysis bag was taken out to complete a labeling process, and a bound compound was sucked from the dialysis bag and then subjected to chromatography with a Sephadex G-25 column to remove the Alexa Fluor® dye. [0067] D. A binding pad: The dimethyltryptamine monoclonal antibody-colloidal gold complex was diluted in a PBS buffer containing 1% of BSA, 50 mg/mL of trehalose and 200 mg/mL of sucrose until was 1/3 of the initial OD value of the complex and then uniformly sprayed on the binding pad at a spray rate of 1.0 μL/cm; and the dimethyltryptamine monoclonal antibody-fluorescein complex was uniformly sprayed on the binding pad at a spray rate of 1.0 μL/cm. [0068] E. Assembly: A dotted sheet base plate, the binding pad, a sample pad and a water-absorbent paper were assembled into a large card and then sliced to obtain a dimethyltryptamine colloidal gold-fluorescence test paper strip.
Example 8
[0069] Preparation of a dimethyltryptamine colloidal gold-fluorescence test paper: The dimethyltryptamine antigen obtained in Example 2 was used as a dimethyltryptamine antigen, the dimethyltryptamine monoclonal antibody obtained in Example 5 was used as a dimethyltryptamine monoclonal antibody, and other preparation processes were the same as those in Example 7.
Example 9
[0070] Preparation of a dimethyltryptamine colloidal gold-fluorescence test paper: The dimethyltryptamine antigen obtained in Example 3 was used as a dimethyltryptamine antigen, the dimethyltryptamine monoclonal antibody obtained in Example 6 was used as a dimethyltryptamine monoclonal antibody, and other preparation processes were the same as those in Example 7.
[0071] Dimethyltryptamine solutions with different concentrations were prepared, the dimethyltryptamine colloidal gold-fluorescence test paper strip prepared in Example 8 was used for carrying out a functional test on samples, the color development intensity of the test line was determined by a colloidal gold colorimetric card, and each concentration of the sample was determined for 5 times to obtain an average value. Results are as shown in Table 1.
TABLE-US-00001 TABLE 1 Color development intensity of a dimethyltryptamine colloidal gold- fluorescence test paper Sample concentration (ng/mL) T line intensity Fluorescence intensity F 0 G9 2301±43 250 G6.5 1395±22 500 G5 846±17 750 G4 513±12 1000 G3.5 311±13 1250 G3 189±12 1500 G2 114±8 1750 G2 69±4 3000 G1 6±1
[0072] Results of the colloidal gold-fluorescence test paper are read by a colorimetric card method. G1 to G10 refer to the color development degree of colloidal gold of the T line, where on the basis of observation with naked eyes, the G1 shows a colorless T line which indicates a strong positive result, and the G10 shows a dark T line which indicates a strong negative result. The fluorescence intensity of samples with different concentrations can be tested by on-machine detection. The fluorescence intensity is negatively correlated with the concentration of the dimethyltryptamine. When the concentration of the dimethyltryptamine in the samples is higher, the fluorescence intensity is lower, and an inversely proportional relationship is achieved in a certain range. From Table 1, it can be seen that the detection concentration of the test paper prepared by the present invention can be as low as 1,000 ng/mL and has high sensitivity. Results can be observed with naked eyes and can also be quantitatively analyzed by detecting the fluorescence intensity.
[0073] The dimethyltryptamine colloidal gold-fluorescence test paper strips prepared in Examples 7-9 were used for carrying out a functional test on 118 clinical urine samples. In the clinical urine samples, 73 cases were negative, and 45 cases were positive. The concentration of the dimethyltryptamine was distributed in 1,000-2,000 ng/mL. Test results are as shown in Table 2.
TABLE-US-00002 TABLE 2 Test results of clinical samples Clinical sample Item Positive Negative Accuracy % Example 7 Positive 45 cases 3 cases 97.5 Negative 0 case 70 cases Example 8 Positive 44 cases 4 cases 95.8 Negative 1 case 69 cases Example 9 Positive 41 cases 6 cases 92.4 Negative 3 cases 67 cases
[0074] From Table 2, it can be seen that the dimethyltryptamine colloidal gold-fluorescence test paper of the present invention has high detection accuracy for clinical samples, and the overall accuracy is greater than 90%.