CN110981875A - Atropine hapten, synthetic method thereof, antigen, antibody and application - Google Patents
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Abstract
The invention discloses an atropine hapten, a synthetic method thereof, an antigen, an antibody and an immunodetection device which is convenient to use, quick, simple and convenient, high in sensitivity and accuracy and capable of measuring atropine on site. An atropine hapten having the structure of formula (I):
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an atropine hapten, a synthetic method thereof, an antigen, an antibody and application thereof.
Background
Atropine is an anticholinergic agent, is an M-receptor blocker, can be used for sedation and analgesia, has certain toxicity, and may cause central nervous poisoning. With the rapid development of economy, people pay more and more attention to the quality of life, so that the health care product has wide market prospect. Driven by the interest, there are some illegal merchants who may rule out the addition of atropine for the relief of gastric ulcers. If the addition amount is excessive, the health of consumers is affected.
At present, the detection methods of atropine at home and abroad mainly comprise High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS) and the like, and instruments and equipment used in the methods are complex in operation, high in cost, high in technical requirement on operators, incapable of displaying results immediately, and not suitable for quick online detection and monitoring of suspected objects by departments such as base layer management and the like. The immunological detection and analysis technology has the advantages of high sensitivity, high specificity, rapidness, simple and convenient operation and the like, is widely applied to the field of drug residue detection, and has many advantages compared with detection methods such as instruments and the like.
An immunological detection method is established and applied to detect the residual atropine quantity, and the key technology is that an antibody with strong specificity and high sensitivity can be obtained, and the aim is to realize the aim under the precondition that a proper atropine hapten is synthesized and prepared. However, at present, no report aiming at atropine hapten and related quick detection products exists in China.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention provides an atropine hapten, a synthetic method thereof, an antigen and an antibody, and an immunoassay device which is convenient to use, quick, simple, convenient, high in sensitivity and accuracy and capable of measuring atropine on site.
In order to solve the technical problems, the invention adopts the following technical scheme:
an atropine hapten having the structure of formula (I):
a method for synthesizing the atropine hapten comprises the following steps:
(1): dissolving nortropine alcohol in DMF, adding alkali, adding 6-bromo-1-hexene, reacting at 60-90 ℃ for several hours, and performing post-treatment to obtain a brown viscous oily substance, namely an intermediate 1, wherein the mass ratio of the nortropine alcohol to the 6-bromo-1-hexene to the alkali is 1: (1-1.5): (1-5);
(2): under the inert gas atmosphere, methyl phenylacetate and ethyl formate generate an intermediate 2 under the action of titanium tetrachloride, wherein the mass ratio of the methyl phenylacetate to the ethyl formate to the titanium tetrachloride is 1: (3-5): (3-5); (3): dissolving the intermediate 1 and the intermediate 2 with toluene, and performing transesterification reaction under the action of sodium methoxide to generate an intermediate 3, wherein the mass ratio of the intermediate 1 to the intermediate 2 is 1: 1;
(4): reducing the intermediate 3 by sodium borohydride, extracting, combining organic phases, evaporating to dryness, and purifying by a column to obtain a light yellow oily liquid, namely an intermediate 4; the mass ratio of the intermediate 3 to the sodium borohydride is 1 (1.1-3);
(5): carrying out reflux reaction on the intermediate 4 under sodium periodate and ruthenium trichloride, evaporating to obtain a solid state, redispersing and dissolving, filtering, evaporating to dryness, and purifying by a column to obtain a black oily liquid, namely atropine hapten, wherein the mass ratio of the intermediate 4, the sodium periodate and the ruthenium trichloride is 1 (1.1-3): (0.3-0.5).
An atropine antigen comprising: an atropine hapten as described above, and a carrier protein conjugated to the atropine hapten. Wherein the carrier protein comprises bovine serum albumin, human serum albumin, chicken egg albumin or hemocyanin.
An atropine antibody, which is an antibody specifically aiming at the atropine antigen. Wherein the atropine antibody is an atropine monoclonal antibody or an atropine polyclonal antibody.
The use of the above-mentioned atropine hapten and/or the above-mentioned atropine antigen and/or the above-mentioned atropine antibody in an immunological test.
An atropine immunoassay device, which comprises the atropine antigen and/or the atropine antibody.
Preferably, but not limitatively, the detection device is a colloidal gold detection device, and specifically, the detection device comprises a test strip and a working solution (i.e. a reaction cup), wherein the test strip comprises a base plate, a sample pad, a nitrocellulose membrane and a water absorption pad, and the sample pad, the nitrocellulose membrane and the water absorption pad are sequentially overlapped on the base plate. The nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line in the test strip is coated with the atropine antigen. The reaction cup contains the atropine antibody marked by colloidal gold.
The preparation method of the atropine colloidal gold detection device comprises the following steps:
(a) preparing a nitrocellulose membrane, wherein a detection line and a quality control line are formed on the nitrocellulose membrane;
the detection line is prepared by coating and scribing atropine coupling antigen capable of being combined with the atropine monoclonal antibody on the nitrocellulose membrane; the quality control line is prepared by coating and scribing a goat anti-mouse antibody which can be combined with the atropine monoclonal antibody. Preferably, a Biodot membrane scribing instrument is adopted for coating and scribing, the specific parameters are that the platform moving speed is 40mm/s, the atropine coupled antigen coating concentration is 2mg/ml, and the unit scribing amount is 1 mu L/cm; the coating concentration of the goat anti-mouse antibody is 1.5mg/ml, and the unit cross-hatching amount is 1 muL/cm.
The atropine conjugate antigen is obtained by the following method: 0.1mmol of atropine hapten is taken to be dissolved in 2mLDMF, and 0.2mmol of DCC and 0.1mmol of NHS are added with stirring. The reaction was magnetically stirred overnight at 4 ℃ and centrifuged to obtain supernatant A, and 140mg of Human Serum Albumin (HSA) was dissolved in 10mL of 0.1mol/L PBS (pH 8.0). Adding 1mL of DMF, stirring and dissolving to prepare solution B, gradually dripping the solution A into the solution B under magnetic stirring, and reacting for 12h at 4 ℃. After centrifugation, the supernatant was dialyzed against physiological saline at 4 ℃ for 3 days, and the dialysate was changed 3 times a day. The whole antigen thus obtained was dispensed into a 0.5mL centrifuge tube at a concentration of 10 mg/mL. Freezing in refrigerator at-20 deg.C for use.
(b) And assembling the test strip, namely overlapping the sample pad, the nitrocellulose membrane and the water absorption pad on the bottom plate in sequence, and pressing the sample pad and the water absorption pad 1-2mm above the nitrocellulose membrane respectively.
(c) Preparing a reaction cup, wherein the reaction cup contains an atropine monoclonal antibody marked by colloidal gold;
the colloidal gold labeled atropine monoclonal antibody is prepared by coupling colloidal gold and the atropine monoclonal antibody.
The detection principle of the atropine colloidal gold device is that a competitive immunochromatography technology is adopted, so that atropine in a sample to be detected, an atropine antigen coated on a detection line and atropine monoclonal antibody marked by colloidal gold are combined in a competitive mode. If the sample to be detected contains atropine, the atropine in the sample to be detected is combined with the colloidal gold labeled atropine monoclonal antibody, so that the combination of the gold labeled atropine monoclonal antibody and the atropine antigen coated on the detection line is inhibited. And obtaining a detection result by comparing the color depth of the detection line and the color depth of the quality control line.
The invention has the beneficial effects that:
the atropine hapten provided by the invention takes nortropine as a raw material, and the proper atropine hapten is prepared and can be more fully exposed to the immune system of animals; the atropine hapten and carrier protein are coupled to prepare artificial antigen, the atropine artificial antigen is used for immunizing animals, and a cell fusion technology is adopted to prepare the monoclonal antibody with high specificity to the atropine.
The immunoassay device for the atropine antigen/antibody, such as a colloidal gold immunochromatography assay device, obtained by the invention can be used for semi-quantitatively detecting the residual atropine amount in a sample by using the color comparison of a detection line and a quality control line in test paper, rapidly and accurately detecting whether the sample contains the atropine in a short time, can meet the field rapid detection requirement of a supervision department and a detection mechanism on the atropine, and has very important practical significance. Compared with the prior art, the invention has the characteristics of convenient use, economy, rapidness, easy manufacture and low cost.
Drawings
FIG. 1 is a schematic structural diagram of an atropine colloidal gold detection device of the present invention, wherein the left side is a test strip and the right side is a reaction cup; in the figure, 1, a water absorption pad, 2, a quality control line, 3, a nitrocellulose membrane, 4, a detection line, 5, a sample pad, 6 and a reaction cup.
FIG. 2 is an atropine hapten mass spectrum.
Detailed Description
The test methods used in the following examples are all conventional methods unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 preparation of atropine hapten
Example 1-1 a method for synthesizing atropine hapten comprising the steps of:
(1): 2.5g of raw materials nortropine, 2.8g of anhydrous potassium carbonate and 27mL of DMF (N, N-dimethylformamide) are added into a 100mL single-neck flask and uniformly stirred, 3.3g of 6-bromo-1-hexene is added, and the mixture is stirred and reacted for 6 hours at 80 ℃ (point TLC plate detection and potassium permanganate color development). After the reaction is finished, cooling to room temperature, carrying out rotary evaporation under reduced pressure by an oil pump, evaporating out the DMF solvent, adding methanol to dissolve, and filtering out insoluble substances. Evaporating methanol, and purifying by column chromatography to obtain brown viscous oily substance, namely an intermediate 1; the specific chemical reaction involved in this step is shown above.
(2): in N2Under an atmosphere, 3.0g of methyl phenylacetate, 2.2g of ethyl formate and 60mL of dichloromethane were added to a 100mL single-neck flask, stirred uniformly and cooled to 0 ℃. 5.67g of titanium tetrachloride was added dropwise during stirring, and 4.5g of triethylamine was added dropwise after completion of the addition of titanium tetrachloride. During which the system turns a blood red color. The dropwise addition was completed, maintained at 0 ℃ for 1h, and then warmed to room temperature for 1h of reaction (the product was close in polarity to the starting material but could be developed with potassium permanganate solution). After the reaction is finished, cooling to 0 ℃, performing extraction quenching reaction by using ice-cold water, extracting for 2-3 times by using ethyl acetate, combining organic phases, and evaporating to dryness to obtain orange liquid, namely an intermediate 2; the specific chemical reaction involved in this step is shown above.
(3): 2.0g of intermediate 1 obtained in the above two steps was mixed with 1.8g of intermediate 2 and 85mL of toluene in a 250mL single-neck flask and stirred well. 0.14g of sodium methoxide is added, and the reaction is refluxed at the external temperature of 130 ℃ for 4 hours. The methanol distilled off was removed with a water separator filled with water. After the reaction is finished, evaporating the solvent to dryness, adding water and 10% sodium hydroxide to adjust the pH value of the solution to be more than 10, extracting for 2-3 times by using ethyl acetate, combining organic phases, evaporating to dryness, and purifying by using a column to obtain a white solid (the impure state is brown yellow), namely an intermediate 3; the specific chemical reaction involved in this step is shown above.
(4): 3.55g of the atropine intermediate 3 obtained in the above step was put into a 250mL single-neck flask, and 80mL of methanol was added thereto and stirred uniformly. After the temperature of the system is reduced to 0 ℃, 0.4g of sodium borohydride is slowly added in batches, a large amount of gas is generated in the process, and the materials cannot be flushed. After the sodium borohydride is added, the system is heated to room temperature, and stirred to react for 8 hours. After the reaction is finished, removing methanol by rotary evaporation, then adding a dilute sodium carbonate solution, extracting for 2-3 times by using ethyl acetate, combining organic phases, drying by distillation, and performing column purification to obtain a light yellow oily liquid, namely an intermediate 4; the specific chemical reaction involved in this step is shown above.
(5): 3.2g of the atropine intermediate 4 obtained in the previous step is put into a 250mL single-neck flask, 25mL of acetonitrile and 38mL of water are added, 2.0g of sodium periodate is added, 0.46g of ruthenium trichloride is added, and 25mL of carbon tetrachloride is added finally. The mixed system is heated to 60 ℃ and refluxed for 2 h. After the reaction was completed, all the solvent was removed by rotary evaporation, and then the obtained solid was scraped off and added to 100mL of methanol and stirred to dissolve the soluble matter as much as possible. Filtering to remove filter residues, evaporating the filtrate to dryness, and purifying by a column to obtain a black oily liquid, namely atropine hapten, wherein the specific chemical reaction formula involved in the step is shown as above, and the mass spectrum identification of the atropine hapten is carried out by EI-MS (positive) m/z: 376.5[ M + H]+As shown in fig. 2.
Example 1-2 a method for synthesizing atropine hapten comprising the steps of:
(1): 5.0g of raw materials nortropine, 11.2g of anhydrous potassium carbonate and 100mL of DMF (N, N-dimethylformamide) are added into a 250mL single-neck flask and uniformly stirred, 8.0g of 6-bromo-1-hexene is added, and the mixture is stirred and reacted for 6 hours at 80 ℃ (point TLC plate detection and potassium permanganate color development). After the reaction is finished, cooling to room temperature, carrying out rotary evaporation under reduced pressure by an oil pump, evaporating out the DMF solvent, adding methanol to dissolve, and filtering out insoluble substances. After evaporation of the methanol to dryness, purification on column afforded a brown viscous oil, intermediate 1.
(2): in N2Under an atmosphere, 6.0g of methyl phenylacetate, 8.8g of ethyl formate and 100mL of dichloromethane were added to a 250mL single-neck flask, stirred uniformly and cooled to 0 ℃. While stirring, 12.0g of titanium tetrachloride was added dropwise, and 10.0g of triethylamine was added dropwise after completion of the addition of titanium tetrachloride. During which the system turns a blood red color. The dropwise addition was completed, maintained at 0 ℃ for 1h, and then warmed to room temperature for 1h of reaction (the product was close in polarity to the starting material but could be developed with potassium permanganate solution). After the reaction is finished, cooling to 0 ℃, performing extraction quenching reaction by using ice-cold water, extracting for 2-3 times by using ethyl acetate, combining organic phases, and evaporating to dryness to obtain orange liquid, namely an intermediate 2.
(3): 4.0g of intermediate 1 obtained in the above two steps was mixed with 3.6g of intermediate 2 and 100mL of toluene in a 250mL single-neck flask and stirred well. 0.3g of sodium methoxide is added, and the mixture is refluxed at the external temperature of 130 ℃ for 4 hours. The methanol distilled off was removed with a water separator filled with water. After the reaction is finished, the solvent is evaporated to dryness, water and 10% sodium hydroxide are added to adjust the pH value of the solution to be more than 10, ethyl acetate is used for extracting for 2-3 times, organic phases are combined, and after evaporation to dryness, the mixture is purified by a column to obtain a white solid (the impure product is brown yellow), namely an intermediate 3.
(4): 7.0g of the atropine intermediate 3 obtained in the above step was put into a 250mL single-neck flask, and 100mL of methanol was added thereto and stirred uniformly. After the temperature of the system is reduced to 0 ℃, 1.6g of sodium borohydride is slowly added in batches, a large amount of gas is generated in the process, and the materials cannot be flushed. After the sodium borohydride is added, the system is heated to room temperature, and stirred to react for 8 hours. And (3) after the reaction is finished, removing methanol by rotary evaporation, adding a dilute sodium carbonate solution, extracting for 2-3 times by using ethyl acetate, combining organic phases, drying by distillation, and performing column purification to obtain a light yellow oily liquid, namely an intermediate 4.
(5): 3.2g of the atropine intermediate 4 obtained in the previous step is put into a 250mL single-neck flask, 25mL of acetonitrile and 38mL of water are added, 4.0g of sodium periodate is added, 0.69g of ruthenium trichloride is added, and 25mL of carbon tetrachloride is added finally. The mixed system is heated to 60 ℃ and refluxed for 2 h. After the reaction is finished, all the solvent is removed by rotary evaporation, and then the obtained solid is scraped and added into 100mL of mixed solution of methanol and water in equal proportion and stirred to dissolve soluble substances as much as possible. Filtering to remove filter residue, evaporating the filtrate to dryness, and purifying with column to obtain black oily liquid, i.e. atropine hapten.
Examples 1-3 a method for synthesizing atropine hapten comprising the steps of:
(1): 10.0g of raw materials, namely nortropine, 54g of anhydrous potassium carbonate and 150mL of DMF (N, N-dimethylformamide) are added into a 500mL single-neck flask and uniformly stirred, 19.3g of 6-bromo-1-hexene is added, and the mixture is stirred and reacted for 6 hours at 80 ℃ (point TLC plate detection, potassium permanganate color development). After the reaction is finished, cooling to room temperature, carrying out rotary evaporation under reduced pressure by an oil pump, evaporating out the DMF solvent, adding methanol to dissolve, and filtering out insoluble substances. After evaporation of the methanol to dryness, purification on column afforded a brown viscous oil, intermediate 1.
(2): in N2Under an atmosphere, 15.0g of methyl phenylacetate, 37g of ethyl formate and 200mL of dichloromethane were added to a 1000mL single-neck flask, stirred uniformly and cooled to 0 ℃. 94.5g of titanium tetrachloride was added dropwise during stirring, and 50.0g of triethylamine was added dropwise after completion of the addition of titanium tetrachloride. During which the system turns a blood red color. The dropwise addition was completed, maintained at 0 ℃ for 1h, and then warmed to room temperature for 1h of reaction (the product was close in polarity to the starting material but could be developed with potassium permanganate solution). After the reaction is finished, cooling to 0 ℃, performing extraction quenching reaction by using ice-cold water, extracting for 2-3 times by using ethyl acetate, combining organic phases, and evaporating to dryness to obtain orange liquid, namely an intermediate 2.
(3): 20.0g of intermediate 1 obtained in the above two steps was mixed with 18g of intermediate 2 and 200mL of toluene in a 500mL single-neck flask and stirred well. After 3.1g of sodium methoxide was added, the reaction was refluxed at 130 ℃ for 4 hours. The methanol distilled off was removed with a water separator filled with water. After the reaction is finished, the solvent is evaporated to dryness, water and 10% sodium hydroxide are added to adjust the pH value of the solution to be more than 10, ethyl acetate is used for extracting for 2-3 times, organic phases are combined, and after evaporation to dryness, the mixture is purified by a column to obtain a white solid (the impure product is brown yellow), namely an intermediate 3.
(4): 7.0g of the atropine intermediate 3 obtained in the above step was put into a 250mL single-neck flask, and 100mL of methanol was added thereto and stirred uniformly. After the temperature of the system is reduced to 0 ℃, 2.24g of sodium borohydride is slowly added in batches, a large amount of gas is generated in the process, and the materials cannot be flushed. After the sodium borohydride is added, the system is heated to room temperature, and stirred to react for 8 hours. And (3) after the reaction is finished, removing methanol by rotary evaporation, adding a dilute sodium carbonate solution, extracting for 2-3 times by using ethyl acetate, combining organic phases, drying by distillation, and performing column purification to obtain a light yellow oily liquid, namely an intermediate 4.
(5): 6.4g of the atropine intermediate 4 obtained in the previous step is put into a 250mL single-neck flask, 50mL of acetonitrile and 72mL of water are added, 11.5 g of sodium periodate is added, 1.54g of ruthenium trichloride is added, and 50mL of carbon tetrachloride is added finally. The mixed system is heated to 60 ℃ and refluxed for 2 h. After the reaction was completed, all the solvent was removed by rotary evaporation, and then the obtained solid was scraped off and added to 100mL of methanol and stirred to dissolve the soluble matter as much as possible. Filtering to remove filter residue, evaporating the filtrate to dryness, and purifying with column to obtain black oily liquid, i.e. atropine hapten.
Example 2 preparation of atropine immunizing antigen
An immunizing antigen was prepared using the atropine hapten prepared in example 1-1. Specifically, 0.1mmol of atropine hapten is taken to be dissolved in 2mLDMF, 0.2mmol of DCC (dicyclohexylcarbodiimide) and 0.15mmol of NHS (N-hydroxysuccinimide) are added with stirring, the mixture is reacted overnight under magnetic stirring at 4 ℃, the supernatant fluid is centrifuged to be A fluid, and 140mg of hemocyanin (KLH) is weighed and dissolved in 10mL of PBS (pH8.0) with the concentration of 0.1 mol/L. Adding DMF1mL, stirring to dissolve to obtain solution B, gradually dripping solution A into solution B under magnetic stirring, and reacting at 4 deg.C for 12 hr. After centrifugation, the supernatant was dialyzed against physiological saline at 4 ℃ for 3 days, and the dialysate was changed 3 times a day. The whole antigen thus obtained was dispensed into a 0.5mL centrifuge tube at a concentration of 1 mg/mL. Freezing in a refrigerator at-20 deg.C.
EXAMPLE 3 preparation of monoclonal antibodies against atropine Immunantigen
Monoclonal antibodies were prepared using the atropine immunizing antigen prepared in example 2. Specifically, atropine immune antigen is used and identified, then BALB/C mice of 4 weeks are immunized, after three times of boosting immunization, blood is taken to measure titer, when serum titer does not rise any more, the mice are immunized by using two doses of antigen without adjuvant, after three days, the mice are killed by removing necks, spleen cells are prepared under aseptic condition, the spleen cells and mouse myeloma cells which grow vigorously are mixed in a 50mL centrifuge tube according to the proportion of 8:1, 30mL serum-free IPMI1640 culture medium is added, centrifugation is carried out at 1100r/min for 5min, supernatant is discarded, cell clusters are loosened by light vibration, and the cells are placed in a water bath at 37 ℃. Slowly adding 50% PEG-4000 mL into cells, dripping within 1min, gently stirring the bottom precipitate, standing for 1min, slowly adding 1mL of serum-free culture medium along the tube wall in the first 30s at constant speed, adding 2mL of serum-free culture medium in the second 30s, rapidly adding 27mL to terminate the fusion process, centrifuging at 1100r/min for 5min, discarding the supernatant, resuspending with HAT selective culture medium, adding into 96-well cell culture plate paved with feeder cells, and culturing at 37 ℃ under the condition of CO2 with volume fraction of 5%. And 7 days later, changing into HT culture solution, when the number of the hybrid cells in the hole reaches more than 300, screening by using an indirect ELISA method, selecting the hole with strong positive, good inhibition effect and vigorous cell growth for limiting dilution and cloning, carrying out cloning culture and detection for more than 3 times, wherein the positive hole cells are the hybrid tumor cells secreting the monoclonal antibody, and carrying out expanded culture on the hybrid tumor cells to prepare the monoclonal antibody.
The atropine-resistant monoclonal antibody is produced by adopting an in vivo induced ascites method. Selecting 4 Kunming mice, injecting liquid paraffin oil into the abdominal cavity of each mouse at a ratio of 0.5mL, and injecting the hybridoma cells into the abdominal cavity after 7 days for 3-5 × 106Ascites was collected after 10 days when the abdomen of the mice had significantly enlarged. The ascites is purified by using an n-octanoic acid-ammonium sulfate precipitation method, and the content of the anti-atropine monoclonal antibody is determined by ultraviolet.
Example 4 preparation of atropine colloidal gold assay device
4.1 preparation of colloidal gold
Taking 1mL of 1% chloroauric acid solution, adding 99mL of ultrapure water to obtain a chloroauric acid solution with the final concentration of 0.01%, heating to boil, quickly adding 1.6mL of 1% trisodium citrate into the boiled chloroauric acid solution at one time, continuously heating until the solution is changed from light yellow to blue black and finally to bright red, continuously heating for 5min after the color is stable, cooling at room temperature, and supplementing water to the original volume.
4.2 preparation of colloidal gold-labeled monoclonal antibody
Adjusting the pH value of the colloidal gold solution to 8.0, uniformly stirring by using a constant-speed stirrer, simultaneously dropwise adding an atropine monoclonal antibody, adding PEG with the equivalent antibody amount after 1h, adding BSA with the equivalent antibody amount after fully reacting for 30min, and continuing stirring for 30min after the addition is finished. Centrifugation is carried out for 30min at 9000rpm to obtain a uniform gold-labeled antibody precipitate, and PNPB is added for re-suspension for later use.
4.3 preparation of colloidal gold detection device
As shown in fig. 1, on the bottom plate, the sample pad, the nitrocellulose membrane sprayed with atropine-BSA (detection line) and goat anti-mouse IgG (quality control line), and the absorbent pad were sequentially lapped and adhered in the same direction; adding the colloidal gold labeled monoclonal antibody into the reaction cup, and freeze-drying.
5.1 pretreatment of the sample: taking 0.1g or 0.1mL of a substance to be detected of health food (hard capsule, soft capsule, tablet, pill, powder, aqueous agent and other dosage forms) into an extraction bottle containing 0.1% acetic acid solution, screwing the bottle cap, shaking for 30 seconds, and mixing uniformly to obtain the liquid to be detected.
5.2, detection: the upper cover of the reagent bottle is unscrewed, 9-10 drops (about 200 mu L) of the solution to be detected are vertically dropped into the reaction cup, and the solution is pumped up and down for 10 times and is mixed evenly. Starting the first-step reaction at 20-40 ℃ and timing for 3 minutes; inserting the test strip into the reaction cup, 20-
The second reaction step is started at 40 ℃ and the time is 3 minutes; and taking the test strip out of the micropore, slightly scraping a sample pad at the lower end of the test strip, and judging the result.
5.3 interpretation of results
(1) Comparison with the result of the verification method
EXAMPLE 6 sensitivity of atropine colloidal gold detection device
Through experiments, the sensitivity of the atropine colloidal gold detection device is as follows: atropine 50 mug/kg.
Example 7 specificity test of atropine colloidal gold detection device
To the negative samples, atropine 50. mu.g/kg, zopiclone 10mg/kg, sulfadimidine 10mg/kg, and clenbuterol 10mg/kg were added, respectively. Experimental results show that only a sample added with atropine can be detected, but a sample added with zopiclone, sulfadimidine and clenbuterol cannot be detected, and the detection device has good specificity on the atropine.
EXAMPLE 8 shelf-Life test of atropine colloidal gold detection device
Three batches of products produced conventionally are respectively used for carrying out quality guarantee period experiments, the products are placed in an indoor room temperature environment to be kept, 12 devices are taken every 1 month, quality control samples are used for detection, negative and 50 mu g/kg, 100 mu g/kg and 150 mu g/kg labeling experiments are respectively carried out, the three times of experiments are repeated, data change is observed, and the quality guarantee period time is inspected. The negative coloration decreased from 13 months, with no significant change in product quality over a 1 year period, thus establishing a shelf life of 1 year.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. An atropine hapten, which has the structure shown in formula (I):
2. a method of synthesizing an atropine hapten according to claim 1, comprising the steps of:
(1): reacting nortropine with 6-bromo-1-hexene under the action of alkali to obtain brown viscous oily substance, namely an intermediate 1;
(2): under the inert gas atmosphere, generating an intermediate 2 by the methyl phenylacetate and ethyl formate under the action of titanium tetrachloride;
(3): dissolving the intermediate 1 and the intermediate 2 with toluene, and carrying out transesterification reaction under the action of sodium methoxide to generate an intermediate 3;
(4): reacting the intermediate 3 with sodium borohydride, extracting, combining organic phases, drying by distillation, and purifying by a column to obtain a light yellow oily liquid, namely an intermediate 4;
(5): and carrying out reflux reaction on the intermediate 4 under sodium periodate and ruthenium trichloride, evaporating to obtain a solid state, dispersing and dissolving, filtering, evaporating a filtrate to dryness, and purifying by a column to obtain a black oily liquid, namely atropine hapten.
3. A method for synthesizing an atropine hapten according to claim 2, wherein in step (1), the quantity ratio of the materials of the nortropine, the 6-bromo-1-hexene and the alkali is 1: (1-1.5): (1-5).
4. A method for synthesizing atropine hapten according to claim 2 or 3, wherein in the step (2), the ratio of the amounts of the substances of methyl phenylacetate, ethyl formate and titanium tetrachloride is 1: (3-5): (3-5).
5. The method for synthesizing atropine hapten according to claim 2, wherein in the step (5), the ratio of the amounts of the intermediate 4, the sodium periodate and the ruthenium trichloride is 1 (1.1-3): (0.3-0.5).
6. An atropine antigen, wherein the atropine antigen comprises: the atropine hapten of claim 1, and a carrier protein conjugated to the atropine hapten.
7. An atropine antibody which is an antibody specific for the atropine antigen of claim 2 or 3.
8. Use of the atropine hapten of claim 1 and/or the atropine antigen of claim 6 and/or the atropine antibody of claim 7 in an immunological assay.
9. An atropine immunoassay device comprising the atropine antigen of claim 7 and/or the atropine antibody of claim 7.
10. The immunoassay device of claim 9, wherein the test device comprises a test strip and a working solution, wherein the detection line in the test strip is coated with the atropine antigen of claim 6; the working solution contains the atropine antibody of claim 7 labeled with colloidal gold.
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| CN113968853A (en) * | 2020-07-22 | 2022-01-25 | 中国农业大学 | Hapten and artificial antigen of atropine alkaloid, and preparation method and application thereof |
| CN114790203A (en) * | 2022-04-25 | 2022-07-26 | 杭州同舟生物技术有限公司 | Scopolamine artificial hapten, scopolamine artificial antigen, and preparation methods and applications thereof |
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