Etomidate hapten and preparation method and application thereof

Abstract

The invention discloses an etomidate hapten as well as a preparation method and application thereof, and relates to the technical field of biochemical engineering, the hapten which almost retains all structures of etomidate and is provided with carboxyl is synthesized from the beginning, and different carrier proteins are further coupled to serve as an artificial immunogen and a coating antigen respectively. The etomidate hapten compound disclosed by the invention is simple and convenient in synthesis method and relatively high in purity, and can be applied to an antigen system for animal immunization, and the prepared anti-etomidate monoclonal antibody can only identify etomidate and does not identify other anaesthetic drugs, is very high in sensitivity, and can be applied to animal immunization. The method can be used for rapid and accurate immunodetection and immunoassay of etomidate.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to an etomidate artificial hapten, an artificial antigen, and a preparation method and application thereof. Background Art

[0002] Etomidate is a non-barbiturate intravenous short-acting hypnotic and one of the commonly used drugs for anesthesia induction. Etomidate has a 30-year history of clinical application and has a strong inhibitory effect on the central nervous system through intravenous injection. Its duration of action can be prolonged accordingly with increasing doses, and it has little effect on the respiratory and circulatory systems. It can cause short-term respiratory depression, a slight decrease in systolic blood pressure, and a slightly increased heart rate. It has no histamine release effect and is suitable for general anesthesia induction and can also be used for short-term surgical anesthesia.

[0003] At present, the detection of etomidate mainly relies on high performance liquid chromatography (HPLC), gas chromatography (GC), thin layer chromatography (TLC), mass spectrometry (MS), etc. However, these methods have the disadvantages of expensive instruments, time-consuming detection, and the need for professional technicians to operate, and cannot meet the requirements of modern detection for rapidity and accuracy.

[0004] Immunoassay can make up for all the above shortcomings. Immunoassay is an analytical method that uses antigen-antibody specific binding reactions to detect various substances (drugs, hormones, proteins, microorganisms, etc.). The key to establishing immunoassay methods for small molecule compounds is to be able to produce antibodies with high affinity and high specificity for small molecule compounds. In order to obtain excellent antibodies, efficient artificial antigens must be prepared first.

[0005] In the prior art, there is no hapten synthesized from scratch for etomidate that retains almost all of the structure of etomidate and has a carboxyl group. The etomidate monoclonal antibody prepared by the present invention can only recognize etomidate and does not recognize other anesthetics. It has good specificity and can be used for the detection and analysis of etomidate. Summary of the invention

[0006] The purpose of the present invention is to provide an artificial etomidate hapten, which is coupled with different carrier proteins as artificial immunogens and coatings, respectively, to prepare anti-etomidate antibodies, which have high sensitivity and good specificity and can be used for rapid analysis and detection of etomidate.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: The present invention provides an artificial hapten of etomidate, whose molecular structure is shown in I: .

[0008] Furthermore, the method for preparing the etomidate artificial hapten comprises the following steps: (1) Dissolve 4-1-bromoethylbenzoic acid in acetonitrile to obtain solution A; (2) Dissolve ethyl imidazole-4-carboxylate in acetonitrile, then add sodium hydroxide to obtain solution B. Slowly add solution A into solution B under temperature control; (3) After the addition of materials is completed, the reaction temperature is set, and after the reaction is completed, the acetonitrile is dried by rotary evaporation; pure water is added to dissolve the product, and ethyl acetate is extracted, the organic layer is discarded, and the pH of the aqueous layer is adjusted with hydrochloric acid. At this time, a white precipitate is precipitated, and the precipitate is filtered and dried to obtain the target hapten; the reaction formula of the method is as follows: .

[0009] Furthermore, in the method for preparing the etomidate artificial hapten, the ratio of the amount of 4-1-bromoethylbenzoic acid in step (1) to the amount of ethyl imidazole-4-carboxylate in step (2) is 1:0.8-1.5; in step (3), the reaction temperature is set at 40-45°C, the reaction time is 6-8h, and the pH is adjusted to 4-5 with hydrochloric acid.

[0010] The present invention provides an etomidate artificial antigen, the molecular structure of which is shown in II: .

[0011] Furthermore, in the etomidate artificial antigen, the protein in structural formula II is at least two of bovine serum albumin, hemocyanin and ovalbumin.

[0012] Furthermore, the preparation method of the etomidate artificial antigen comprises the following steps: (1) Take the artificial hapten etomidate and dissolve it in N,N-dimethylformamide to prepare solution C; (2) Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide separately, mix them together and dissolve them in N,N-dimethylformamide to prepare solution D; (3) Solution D is added dropwise to solution C under ice bath conditions, and then stirred at room temperature overnight. After terminating the reaction, the hapten-activated ester E is obtained.

[0013] Furthermore, in the method for preparing the etomidate artificial antigen, in step (2), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:0.8-1.5.

[0014] Furthermore, the method for preparing the etomidate artificial antigen comprises the following steps: (1) Take hapten activated ester E, add it to PBS containing protein 1, and react overnight at 4°C to obtain reaction solution F; (2) The reaction solution F is placed in a dialysis bag and fully dialyzed with PBS to obtain an artificial antigen, i.e., an immunogen; (3) Take hapten activated ester E, add it to PBS containing protein 2, and react overnight at 4°C to obtain reaction solution G; (4) Place the reaction solution G into a dialysis bag and dialyze it thoroughly with PBS to obtain the artificial antigen, i.e., the coating antigen.

[0015] Furthermore, in the method for preparing the etomidate artificial antigen, the mass ratio of the hapten activated ester to the protein 1 or protein 2 in step (1) and step (3) is 1:3-9; the PBS concentration is 0.01 mol / L, and the pH is 7.4.

[0016] Furthermore, the use of etomidate hapten or etomidate artificial antigen in the preparation of anti-etomidate antibodies.

[0017] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The present invention synthesizes a hapten from scratch that retains almost the entire structure of etomidate and has a carboxyl group, and further couples it with different carrier proteins as artificial immunogens and coatings, thereby preparing etomidate detection antibodies, which have high sensitivity and good specificity and can be used for rapid analysis and detection of etomidate.

[0018] 2. The etomidate monoclonal antibody prepared by the present invention can only recognize etomidate, and cannot recognize other types of anesthetics; this indicates that the etomidate monoclonal antibody of the present invention has good specificity and can be applied to the detection and analysis of etomidate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 is the chemical structural formula of the etomidate artificial hapten of the present invention; Figure 2 is the chemical structural formula of the etomidate artificial antigen of the present invention, wherein the protein is at least two of bovine serum albumin, hemocyanin and ovalbumin.

[0021] Figure 3 This is the reaction formula for preparing the etomidate artificial hapten of the present invention. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods, but the protection scope of the present invention is not limited thereto.

[0023] Example 1

[0024] This embodiment provides a method for preparing an etomidate artificial antigen, comprising the following steps: 1. Preparation of hapten See also Figure 1 , Figure 3 As shown, in this example, 4-1-bromoethylbenzoic acid is used as the starting material, and a hapten having the complete structure of etomidate is prepared through one step of chemical synthesis, and its molecular weight is 288. The specific preparation steps are as follows: (1) Take 2.3 g of 4-1-bromoethylbenzoic acid and dissolve it in 10 mL of acetonitrile to obtain solution A; (2) Take 0.8 g of ethyl imidazole-4-carboxylate, add it into a reaction bottle and dissolve it with 20 mL of acetonitrile, then add 1.0 g of sodium hydroxide to obtain solution B; under temperature control, slowly add solution A into solution B; (3) After the addition is completed, the temperature is raised to 40°C and the reaction is carried out for 6 hours. After the reaction is completed, the acetonitrile is dried by rotary evaporation. 20 mL of pure water is added to dissolve the product, and ethyl acetate is extracted twice. The organic layer is discarded, and the pH of the aqueous layer is adjusted to 4 with 1 mol / L hydrochloric acid. At this time, a white precipitate is precipitated. The precipitate is filtered and dried to obtain the target hapten.

[0025] 2. Preparation of hapten-activated esters The specific preparation steps are as follows: (1) In a 5 mL glass bottle, take 14.5 mg of hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare Solution C; (2) Weigh 10 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 6 mg of N-hydroxysuccinimide respectively, mix them together, and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution D; (3) Add solution D dropwise into solution C under ice bath conditions, and then stir at room temperature overnight. After the reaction is terminated, the hapten-activated ester E is obtained; 3. Synthesis of artificial antigens See also Figure 2 As shown, the specific preparation steps are as follows: (1) Take 4 mg of hapten-activated ester E and add it to 5 mg of hemocyanin dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution F; (2) The reaction solution F was placed in a dialysis bag and dialyzed with a 0.01 mol / L PBS solution to obtain an artificial antigen (immunogen); (3) Take 7.5 mg of hapten-activated ester E and add it to 33 mg of BSA dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution G; (4) Place the reaction solution G into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS solution to obtain the artificial antigen (coating antigen).

[0026] Example 2

[0027] This embodiment provides a method for preparing an etomidate artificial antigen, comprising the following steps: 1. Preparation of hapten See also Figure 1 , Figure 3 As shown, in this example, 4-1-bromoethylbenzoic acid is used as the starting material, and a hapten having the complete structure of etomidate is prepared through one step of chemical synthesis, and its molecular weight is 288. The specific preparation steps are as follows: (1) Take 2.3 g of 4-1-bromoethylbenzoic acid and dissolve it in 10 mL of acetonitrile to obtain solution A; (2) Take 1.5 g of ethyl imidazole-4-carboxylate, add it into a reaction bottle and dissolve it with 20 mL of acetonitrile, then add 1.0 g of sodium hydroxide to obtain liquid B; under temperature control, slowly add liquid A into liquid B; (3) After the addition is completed, the temperature is raised to 42°C and the reaction is carried out for 7 hours. After the reaction is completed, the acetonitrile is dried by rotary evaporation. 20 mL of pure water is added to dissolve the product, and ethyl acetate is extracted twice. The organic layer is discarded, and the pH of the aqueous layer is adjusted to 4.5 with 1 mol / L hydrochloric acid. At this time, a white precipitate is precipitated. The precipitate is filtered and dried to obtain the target hapten.

[0028] 2. Preparation of hapten-activated esters The specific preparation steps are as follows: (1) In a 5 mL glass bottle, take 29 mg of hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare Solution C; (2) Weigh 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 12 mg of N-hydroxysuccinimide respectively, mix them together, and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution D; (3) Add solution D dropwise into solution C under ice bath conditions, and then stir at room temperature overnight. After the reaction is terminated, the hapten-activated ester E is obtained; 3. Synthesis of artificial antigens See also Figure 2 As shown, the specific preparation steps are as follows: (1) Take 8 mg of hapten-activated ester E and add it to 30 mg of hemocyanin dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution F; (2) The reaction solution F was placed in a dialysis bag and dialyzed with a 0.01 mol / L PBS solution to obtain an artificial antigen (immunogen); (3) Take 15 mg of hapten-activated ester E and add it to 50 mg of ovalbumin dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution G; (4) Place the reaction solution G into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS solution to obtain the artificial antigen (coating antigen).

[0029] Example 3

[0030] This embodiment provides a method for preparing an etomidate artificial antigen, comprising the following steps: 1. Preparation of hapten See also Figure 1 , Figure 3 As shown, in this example, 4-1-bromoethylbenzoic acid is used as the starting material, and a hapten having the complete structure of etomidate is prepared through one step of chemical synthesis, and its molecular weight is 288. The specific preparation steps are as follows: (1) Take 2.5 g of 4-1-bromoethylbenzoic acid and dissolve it in 10 mL of acetonitrile to obtain solution A; (2) Take 1.5 g of ethyl imidazole-4-carboxylate, add it into a reaction bottle and dissolve it with 20 mL of acetonitrile, then add 1.0 g of sodium hydroxide to obtain liquid B; under temperature control, slowly add liquid A into liquid B; (3) After the addition of materials, heat to 45°C and react for 8 hours. After the reaction is complete, spin dry the acetonitrile. Add 20 mL of pure water to dissolve the product, extract twice with ethyl acetate, discard the organic layer, and adjust the pH of the aqueous layer to 5 with 1 mol / L hydrochloric acid. At this time, a white precipitate is precipitated. Filter and dry the precipitate to obtain the target hapten.

[0031] 2. Preparation of hapten-activated esters The specific preparation steps are as follows: (1) In a 5 mL glass bottle, take 30 mg of hapten and dissolve it in 1 mL of N,N-dimethylformamide to prepare solution C; (2) Weigh 22 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 14 mg of N-hydroxysuccinimide respectively, mix them together, and dissolve them in 0.5 mL of N,N-dimethylformamide to prepare solution D; (3) Add solution D dropwise into solution C under ice bath conditions, and then stir at room temperature overnight. After the reaction is terminated, the hapten-activated ester E is obtained; 3. Synthesis of artificial antigens See also Figure 2 As shown, the specific preparation steps are as follows: (1) Take 8 mg of hapten activated ester E and add it to 64 mg of bovine serum albumin dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution F; (2) The reaction solution F was placed in a dialysis bag and dialyzed with a 0.01 mol / L PBS solution to obtain an artificial antigen (immunogen); (3) Take 15 mg of hapten-activated ester E and add it to 45 mg of ovalbumin dissolved in PBS (0.01 mol / L, pH = 7.4), and react overnight at 4°C to obtain reaction solution G; (4) Place the reaction solution G into a dialysis bag and dialyze it thoroughly with 0.01 mol / L PBS solution to obtain the artificial antigen (coating antigen).

[0032] Example 4

[0033] This embodiment provides a method for preparing an etomidate monoclonal antibody, comprising the following steps: 1. Animal immunization (1) Balb / C female mice, about 7 weeks old, were selected as immunized animals and numbered 1, 2, 3, and 4. Immunization was performed by subcutaneous injection at multiple points on the back of the neck.

[0034] (2) Immunization procedure: Primary immunization: To improve the immunogenicity of the antigen, an immunogen emulsified with Freund's complete adjuvant is used. Equal amounts of the immunogen and Freund's complete adjuvant are mixed and emulsified before use. The dose is 0.1 mg. Reinforcement immunization: Reinforcement immunization is performed on the 21st and 42nd days after the primary immunization. The emulsification method is the same as the primary immunization, and the dose is 0.05 mg. Thereafter, reinforcement immunization is performed every 21 days, for a total of 5 immunizations.

[0035] 2. Analysis of antiserum effect (1) Starting from the third booster immunization, on the 8th day after each immunization, 30 μL of blood was collected from the mouse tail, centrifuged to obtain antiserum, and stored at -20°C for later use.

[0036] (2) The titer and specificity of the antiserum were determined by indirect enzyme-linked immunosorbent assay. The steps are as follows: S1. Coating: Dilute 5 mg / mL of coating antigen 10,000 times with coating buffer and add 100 μL / well to the ELISA plate as a blank control group, incubate overnight in a 4°C refrigerator, and then wash once with PBST (phosphate buffered saline 0.05% (V / V) Tween20 washing solution).

[0037] S2. Blocking: Pour out the liquid in the wells, wash the plate once, add 200μL of washing solution to each well, spin dry, add 200μL of blocking solution (1% BSA-PBS) to each well, block at 37℃ for 2h, spin dry, and place in a 4℃ refrigerator for later use.

[0038] S3. Sample addition: Add 50 μL of standard diluent, diluted enzyme-labeled secondary antibody and diluted serum to each control well; add 50 μL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum to each sample well; shake and mix, react at 37°C for 30 minutes, wash the plate 3 times with a plate washer, add 200 μL of washing solution to each well, and spin dry.

[0039] S4. Color development: Add 50 μL of color development solution A and B to each well, place in a 37°C oven for color development for 15 minutes, and then add 50 μL of stop solution (1 moL / L H2SO4) to each well.

[0040] S5. Determination: Use an enzyme-linked immunosorbent assay to determine the absorbance of each well at A450nm. Select the antiserum dilution multiple with an absorbance value in the range of 1.5 to 2.0 as the antiserum titer. The effect of the antiserum is determined by its inhibition rate. Under the same drug concentration, the higher the inhibition rate, the higher the sensitivity of the antibody to the drug.

[0041] 3. Cell fusion and screening of positive hybridomas (1) Resuscitation of myeloma cells: Take the myeloma cells out of liquid nitrogen and quickly thaw them in a 37°C water bath. Centrifuge at 1000 rpm for 5 min after thawing. Pour off the supernatant in a clean bench and add about 1 mL of complete culture medium to the cell pellet. Blow away the cells, take out the cells with a pipette, mix with the complete culture medium, and place in a 25 cm 2 The cells are then grown in culture bottles and expanded to 4 to 6 bottles, during which the liquid needs to be changed several times. When the cells in each culture bottle cover the bottom, they can be used for cell fusion.

[0042] (2) Preparation of feeder cells: Balb / C mice were killed by cervical dislocation 1 day before cell fusion, and after being soaked in 75% alcohol for 5 minutes, they were moved to a clean bench for dissection. The abdomen was cut open, the abdominal skin was peeled off, the peritoneum was cut open, the spleen was removed, and moved to a 9 cm culture dish. DMEM basal culture medium was added, and the rinsing liquid was repeatedly aspirated and rinsed with a syringe. The rinsing liquid was transferred to a 50 mL centrifuge tube and centrifuged at 1200 r / min for 5 minutes. The supernatant was discarded, and the cell pellet at the bottom was resuspended with complete culture medium and added to a 96-well cell culture plate, 100 μL per well.

[0043] (3) Preparation of spleen cells: Bleed the orbits of Balb / c mice that have been immunized multiple times and have passed blood tests, collect the serum, and then soak in 75% alcohol for 5 minutes for disinfection, and move to the clean bench for dissection. Remove the spleen aseptically, rinse with DMEM basal culture medium, and place in a culture dish for later use. Use a disposable syringe to absorb the culture medium, hold the removed spleen with tweezers in the left hand, insert the syringe into the spleen with the right hand, and slowly inject the culture medium to wash out the cells in the spleen. Repeat until the spleen turns from dark red to colorless and transparent, and discard the spleen. Collect the mixed culture medium into a 50mL centrifuge tube, seal it, and centrifuge it at 1200r / min for 6 minutes. After centrifugation, discard the supernatant and set aside.

[0044] (4) Cell fusion: Mix the myeloma cells and immune spleen cells after centrifugation in a ratio of about 1:6, add 25 mL of DMEM basal medium, seal the tube, and centrifuge at 1500 r / min for 8 minutes. Discard the supernatant of the myeloma cells and immune spleen cells mixed by centrifugation, and use a gun to remove the excess medium with the mouth of the centrifuge tube facing downward. Loosen the precipitated cells with your fingers, place the centrifuge tube in 37°C warm water, use the gun tip to absorb 1 mL of PEG preheated to 37°C, and slowly add PEG to the precipitated cells within 1 minute. After adding each drop of PEG, gently stir with the gun tip to mix it. Let it stand for 1 minute, preheat the complete medium, add 10 mL of PEG within 2 minutes, and add it along the wall with gentle stirring to mix the PEG. After sealing the centrifuge tube, centrifuge at 1000 r / min for 10 minutes, pour off the supernatant, add HAT complete medium, use a bent pipette to gently aspirate the liquid, stir gently, and evenly add it to the 10 96-well culture plates containing feeder cells prepared the day before. Keep the volume of HAT medium containing feeder cells and HAT medium containing fused cells equal in each well.

[0045] (5) Screening of positive hybridomas: Change the medium with HAT culture medium once within 3 to 4 days after fusion. After 7 days, extract the supernatant in the multi-well culture plate and detect the specific antibodies in the culture medium by indirect ELISA. Select the positive hybridoma cells with high titer and strong drug inhibition. Screen the positive wells with the best fusion effect and mark them. Under sterile conditions, pick the cells growing in clusters in the positive wells with a microscope and transfer them to a 96-well culture plate pre-plated with feeder cells. Each original well is cloned into 96 wells. After the cells adhere to the wall and cover 1 / 4 of the bottom of the well, take the supernatant and test it with icELISA. The titer and inhibition rate are also used as the measurement indicators. The strong positive cells are subcloned by limiting dilution method. Repeat this process for 3 to 4 rounds (note that the positive well cells picked out in each round need to be expanded and cultured, and then frozen for future use) until each well on each plate is positive and the titer and inhibition are similar after detection. At this point, the hybridoma cell line is successfully established, and a hybridoma cell line that can stably secrete uniform antibodies is obtained. Single cell clones were picked and those that tested positive were transferred to 24-well cell culture plates or cell culture dishes for expansion and cryopreservation in time.

[0046] 4. Large-scale preparation of monoclonal antibodies (1) After obtaining hybridoma cell clones that secrete specific monoclonal antibodies, monoclonal antibodies are usually prepared in large quantities by in vitro culture and in vivo monoclonal antibody induction methods. Liquid paraffin is injected into the peritoneal cavity of more than ten Balb / c mice over 8 weeks old in advance, with a dose of 0.5 mL / mouse. Hybridoma cells are injected into the peritoneal cavity of the mice 1 to 2 weeks later.

[0047] (2) After inoculation of cells, the condition of the mice was observed every day. In particular, starting from the 7th day, the abdominal cavity of the mice would swell. Aseptically collect ascites with a disposable syringe before the mice died. The collected ascites was centrifuged at 12000r / min for 10min to remove the upper fat and lower fibrin. The middle layer was collected and its potency and inhibition rate were determined by icELISA method. After purification, it was stored at -20℃ for future use.

[0048] Example 5

[0049] This embodiment provides a method for determining etomidate by indirect competitive enzyme-linked immunosorbent assay (icELISA), which specifically comprises the following steps: 1. Coating: Dilute 5 mg / mL of coating antigen 10,000 times with coating buffer, and make a blank control group. Add 100 μL / well to the ELISA plate and incubate it in a 4°C refrigerator overnight. Then wash once with PBST (phosphate buffer 0.05% (V / V) and Tween20 washing solution.

[0050] 2. Blocking: Pour off the liquid in the wells, wash the plate once, add 200μL of washing solution to each well, spin dry, add 200μL of blocking solution to each well, block at 37℃ for 2h, spin dry, and place in a 4℃ refrigerator for later use.

[0051] 3. Sample addition: Add 50 μL of standard diluent, diluted enzyme-labeled secondary antibody and diluted serum to each control well; add 50 μL of diluted standard, diluted enzyme-labeled secondary antibody and diluted serum to each sample well; shake and mix, react at 37°C for 30 minutes, wash the plate 3 times with a plate washer, add 200 μL of washing solution to each well, and spin dry.

[0052] 4. Color development: Add 50 μL of color development solution A and B to each well, place in a 37°C oven for 15 minutes, and then add 50 μL of stop solution (1 moL / L H2SO4) to each well.

[0053] 5. Determination: Use an enzyme-linked immunosorbent assay (ELISA) to determine the absorbance of each well at A450nm.

[0054] 6. Calculation: Calculate the IC of the inhibition curve using the GraphPad Prism10.0 fitting module 50 value.

[0055] Etomidate standard solutions of 24.3 ng / mL, 8.1 ng / mL, 2.7 ng / mL, 0.9 ng / mL, and 0.3 ng / mL were prepared to establish a standard curve for the indirect enzyme-linked immunosorbent assay. 50 =2.8ng / mL, and the linear detection range is 0.3~24.3ng / mL.

[0056] Experimental example The antibody cross-reaction rate was determined experimentally. According to the optimal coating antigen concentration and the optimal antiserum dilution multiple obtained in Example 3, an indirect competitive ELISA experiment was performed with etomidate, methaqualone, cocaine, etc. as competitive standards to detect the specificity of the etomidate monoclonal antibody. The half inhibitory concentration (IC 50 ) and cross-reaction rate (CR) values ​​are listed in Table 1.

[0057] Table 1 drug <![CDATA[IC 50 (ng / mL)]]> Cross-reaction rate (%) Etomidate 2.8 100 Methaqualone >500 <1 cocaine >500 <1 The experimental results show that the etomidate monoclonal antibody only recognizes etomidate and does not recognize other anesthetics, indicating that the etomidate monoclonal antibody of the present invention has good specificity and can be applied to the detection and analysis of etomidate.

[0058] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

[0059] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to only specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An artificial hapten of etomidate, characterized in that: Its molecular structure is shown in I: 。 2. The method for preparing the artificial hapten of etomidate according to claim 1, characterized in that: The method comprises the following steps: (1) Dissolve 4-1-bromoethylbenzoic acid in acetonitrile to obtain solution A; (2) Dissolve ethyl imidazole-4-carboxylate in acetonitrile, then add sodium hydroxide to obtain solution B. Slowly add solution A into solution B under temperature control; (3) After the addition of materials is completed, the reaction temperature is set, and after the reaction is completed, the acetonitrile is dried by rotary evaporation; pure water is added to dissolve the product, and ethyl acetate is extracted, the organic layer is discarded, and the pH of the aqueous layer is adjusted with hydrochloric acid. At this time, a white precipitate is precipitated, and the precipitate is filtered and dried to obtain the target hapten; the reaction formula of the method is as follows: 。 3. The method for preparing the artificial hapten of etomidate according to claim 2, characterized in that: The molar ratio of 4-1-bromoethylbenzoic acid in step (1) to ethyl imidazole-4-carboxylate in step (2) is 1:0.8-1.5; in step (3), the reaction temperature is set at 40-45°C, the reaction time is 6-8h, and the pH is adjusted to 4-5 with hydrochloric acid.

4. An etomidate artificial antigen, characterized in that: Its molecular structure is shown in II: 。 5. The etomidate artificial antigen according to claim 4, characterized in that The proteins in structural formula II are at least two of bovine serum albumin, hemocyanin and ovalbumin.

6. The method for preparing the etomidate artificial antigen according to claim 4, characterized in that: The preparation steps are as follows: (1) Take the artificial hapten etomidate and dissolve it in N,N-dimethylformamide to prepare solution C; (2) Weigh 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide separately, mix them together and dissolve them in N,N-dimethylformamide to prepare solution D; (3) Solution D is added dropwise to solution C under ice bath conditions, and then stirred at room temperature overnight. After terminating the reaction, the hapten-activated ester E is obtained.

7. The method for preparing the etomidate artificial antigen according to claim 6, characterized in that: In step (2), the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to N-hydroxysuccinimide is 1:0.8-1.

5.

8. The method for preparing the etomidate artificial antigen according to claim 6, characterized in that: The method comprises the following steps: (1) Take hapten activated ester E, add it to PBS containing protein 1, and react overnight at 4°C to obtain reaction solution F; (2) The reaction solution F is placed in a dialysis bag and fully dialyzed with PBS to obtain an artificial antigen, i.e., an immunogen; (3) Take hapten activated ester E, add it to PBS containing protein 2, and react overnight at 4°C to obtain reaction solution G; (4) Place the reaction solution G into a dialysis bag and dialyze it thoroughly with PBS to obtain the artificial antigen, i.e., the coating antigen.

9. The method for preparing the etomidate artificial antigen according to claim 6, characterized in that: In step (1) and step (3), the mass ratio of hapten activated ester to protein 1 or protein 2 is 1:3-9; the concentration of PBS is 0.01 mol / L, and the pH is 7.

4.

10. Use of an artificial etomidate hapten according to claim 1 or an artificial etomidate antigen according to claim 4 in the preparation of anti-etomidate antibodies.

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