Methyl parathion hapten, sibutramine hapten, methyl parathion-sibutramine artificial antigen and antibody, detection kit

Abstract

The present invention provides a methyl parathion hapten, a sibutramine hapten, a methyl parathion-sibutramine artificial antigen and antibody, and a detection kit, relating to the technical field of food safety detection. The polyclonal antibody obtained by immunization with the artificial antigen not only has high sensitivity but also can simultaneously identify both methyl parathion and sibutramine, thus solving the technical problem of the lack of a highly sensitive and rapid method for the simultaneous detection of methyl parathion and sibutramine in the prior art. The half inhibitory concentration (IC50) of the antibody for methyl parathion is 100%. 50 ) was 4.59 ng / mL, the quantitative detection range was 2.89 ng / mL-7.28 ng / mL, and the detection limit was 2.21 ng / mL; the half-inhibitory concentration (IC 50 ) was 59.76 ng / mL, the quantitative detection range was 37.65 ng / mL-94.87 ng / mL, and the detection limit was 28.73 ng / mL.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, in particular to a methyl parathion hapten, a sibutramine hapten, a methyl parathion-sibutramine artificial antigen and antibody, and a detection kit. Background Art

[0002] Sibutramine, a norepinephrine inhibitor, contributes to weight loss by suppressing the central nervous system and reducing appetite. However, it can also cause adverse reactions such as anorexia, insomnia, and liver dysfunction. It can even lead to serious consequences such as cardiovascular and cerebrovascular diseases, neurological disorders, and damage to the nervous system. Therefore, the production, sale, and use of sibutramine preparations and their APIs are prohibited.

[0003] Methyl parathion is a broad-spectrum organophosphorus insecticide widely used to control pests in commercial crops such as fruit trees and tea. However, it is highly toxic, and residues in fruit can enter the human body through the food chain, causing severe toxic effects on the nervous and immune systems. Consequently, methyl parathion has been listed as a banned pesticide. However, due to its significant insecticide efficacy, its abuse continues.

[0004] Methyl parathion and sibutramine are both small molecules with no immune activity. They must be connected to protein molecules to induce an immune response in the body. Therefore, there is an urgent need for a reliable methyl parathion-sibutramine artificial antigen and its preparation method.

[0005] Currently, the main methods for detecting methyl parathion and sibutramine include instrumental analysis methods represented by high-performance liquid chromatography and capillary electrophoresis, and immunoassay methods represented by enzyme-linked immunosorbent assay (ELISA). Although instrumental analysis methods can accurately detect samples, they require complex sample pretreatment, expensive instruments, and long detection times, making them only suitable for quantitative laboratory analysis. Immunoassay methods are simple to operate, low in cost, and fast in detection speed, making them suitable for rapid, on-site, large-scale testing of samples and can better meet the testing needs of growers, enterprises, and regulatory authorities.

[0006] Currently, there are many ELISA methods for detecting methyl parathion, but fewer for sibutramine. Based on the ELISA method for methyl parathion, a polyclonal antibody capable of simultaneously recognizing both methyl parathion and sibutramine was prepared, and a corresponding rapid ELISA analysis method was established. This allows for the simultaneous detection of both methyl parathion and sibutramine in samples by ELISA, which is of great practical significance.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] One of the purposes of the present invention is to provide a methyl parathion hapten which can be coupled with a carrier protein to obtain a novel artificial antigen structure.

[0009] The second object of the present invention is to provide a method for preparing methyl parathion hapten, which has a simple process and a high success rate.

[0010] A third object of the present invention is to provide a sibutramine hapten.

[0011] A fourth object of the present invention is to provide a method for preparing sibutramine hapten, which has a simple process and a high success rate.

[0012] A fifth object of the present invention is to provide a methyl parathion-sibutramine artificial antigen, wherein the polyclonal antibody obtained from the artificial antigen can simultaneously recognize methyl parathion and sibutramine with good recognition ability.

[0013] The sixth object of the present invention is to provide a methyl parathion-sibutramine polyclonal antibody that can simultaneously recognize methyl parathion and sibutramine with good specificity and high sensitivity.

[0014] The seventh object of the present invention is to provide an application of a methyl parathion-sibutramine polyclonal antibody, which solves the technical problem that the prior art lacks a highly sensitive and rapid method for the simultaneous detection of methyl parathion and sibutramine.

[0015] An eighth object of the present invention is to provide a detection kit for methyl parathion and sibutramine, which can simultaneously identify two drugs, methyl parathion and sibutramine, with good specificity and high sensitivity.

[0016] The ninth object of the present invention is to provide an application of a detection kit for methyl parathion and sibutramine, which solves the technical problem of the lack of a highly sensitive and rapid method for the simultaneous detection of methyl parathion and sibutramine in the prior art.

[0017] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0018] In a first aspect, a methyl parathion hapten is provided, wherein the structural formula of the methyl parathion hapten is shown in formula (I):

[0019]

[0020] In a second aspect, a method for preparing a methyl parathion hapten comprises the following steps:

[0021] O,O-diethylthiophosphoryl chloride reacts with p-hydroxybenzoic acid to obtain the methyl parathion hapten.

[0022] In a third aspect, a sibutramine hapten is provided, wherein the structural formula of the sibutramine hapten is shown in formula (II):

[0023]

[0024] A fourth aspect is a method for preparing a sibutramine hapten, comprising the following steps:

[0025] 1-(4-chlorophenyl)cyclobutanecarbonitrile reacts under the action of isobutylmagnesium bromide and sodium borohydride to obtain the sibutramine hapten.

[0026] In a fifth aspect, a methyl parathion-sibutramine artificial antigen is obtained by coupling the above-mentioned methyl parathion hapten and the above-mentioned sibutramine hapten with a carrier protein through the active ester method.

[0027] Furthermore, the carrier protein includes bovine serum albumin and chicken ovalbumin.

[0028] In a sixth aspect, a methylparathion-sibutramine polyclonal antibody is obtained by immunizing an animal with any of the artificial antigens described above.

[0029] In a seventh aspect, a use of the above-mentioned methyl parathion-sibutramine polyclonal antibody in immunoassay of methyl parathion and sibutramine.

[0030] In an eighth aspect, a detection kit for methyl parathion and sibutramine is provided, wherein the detection kit comprises the methyl parathion-sibutramine polyclonal antibody described above.

[0031] In a ninth aspect, a use of the above-mentioned detection kit in immunoassay of methyl parathion and sibutramine.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] The methyl parathion hapten provided by the invention can obtain a novel artificial antigen structure after being coupled with a carrier protein.

[0034] The preparation method of the methyl parathion hapten provided by the invention has simple process and high success rate.

[0035] The sibutramine hapten provided by the present invention can obtain a novel artificial antigen structure after being coupled with a carrier protein.

[0036] The preparation method of sibutramine hapten provided by the present invention has simple process and high success rate.

[0037] The methyl parathion-sibutramine artificial antigen provided by the present invention is a novel artificial antigen structure. The polyclonal antibody obtained from the artificial antigen can simultaneously recognize methyl parathion and sibutramine with good recognition ability.

[0038] The methyl parathion-sibutramine polyclonal antibody provided by the present invention can simultaneously identify both methyl parathion and sibutramine drugs, has good specificity and high sensitivity, provides core raw materials for establishing a specific immunoassay method for methyl parathion and sibutramine, and has broad development prospects.

[0039] The application of the methyl parathion-sibutramine polyclonal antibody provided by the present invention solves the technical problem that there is a lack of a highly sensitive and rapid method for simultaneously detecting methyl parathion and sibutramine in the prior art.

[0040] The detection kit for methyl parathion and sibutramine provided by the present invention can simultaneously identify the two drugs methyl parathion and sibutramine, and has good specificity and high sensitivity.

[0041] The application of the detection kit for methyl parathion and sibutramine provided by the present invention solves the technical problem of the lack of a highly sensitive and rapid method for simultaneously detecting methyl parathion and sibutramine in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1 A schematic diagram of the synthesis process of methyl parathion hapten provided in one embodiment of the present invention;

[0044] Figure 2 A schematic diagram of the synthesis process of sibutramine hapten provided in one embodiment of the present invention;

[0045] Figure 3 The effect of combining methyl parathion first and then sibutramine at different concentrations on the inhibition rate provided by one embodiment of the present invention;

[0046] Figure 4 The effect of combining sibutramine first and then combining methyl parathion at different concentrations on the inhibition rate provided by one embodiment of the present invention;

[0047] Figure 5 This is the UV scanning identification curve of the methyl parathion-sibutramine artificial antigen obtained in Example 3 of the present invention;

[0048] Figure 6 The indirect competition ELISA standard curve of the methyl parathion polyclonal antibody obtained in Example 5 of the present invention;

[0049] Figure 7 This is the standard curve of the sibutramine polyclonal antibody indirect competition ELISA obtained in Example 5 of the present invention. DETAILED DESCRIPTION

[0050] The technical solutions of the present invention are described clearly and completely below with reference to the embodiments. It is obvious that the embodiments described are only a portion of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0051] According to a first aspect of the present invention, there is provided a methyl parathion hapten, the structural formula of which is shown in formula (I):

[0052]

[0053] The methyl parathion hapten of the present invention is named as 4-(diethoxythiophosphate)benzoic acid using a systematic nomenclature.

[0054] The methyl parathion hapten provided by the present invention can obtain a novel artificial antigen structure after being coupled with a carrier protein.

[0055] According to a second aspect of the present invention, there is provided a method for preparing the above-mentioned methyl parathion hapten, comprising the following steps:

[0056] O,O-diethylthiophosphoryl chloride reacts with p-hydroxybenzoic acid to produce methyl parathion hapten.

[0057] In the present invention, p-hydroxybenzoic acid is linked to O,O-diethylthiophosphoryl chloride to form methyl parathion hapten (4-(diethoxythiophosphorothioate)benzoic acid), which has the structure shown in formula (I).

[0058] In the present invention, O,O-diethylthiophosphoryl chloride is added dropwise to a mixture of p-hydroxybenzoic acid, potassium hydroxide and methanol, stirred for reaction at 0° C., and then filtered and purified to obtain methyl parathion hapten.

[0059] The preparation method of the methyl parathion hapten provided by the invention has simple process and high success rate.

[0060] A typical preparation method of methyl parathion hapten is shown in Figure 1 , including the following steps:

[0061] (1) O,O-diethylthiophosphoryl chloride is added dropwise to a mixture of p-hydroxybenzoic acid, potassium hydroxide, and methanol, stirred, filtered, and the solvent is removed under reduced pressure;

[0062] (2) Add distilled water and extract with chloroform several times, then acidify the liquid with hydrochloric acid and extract with chloroform several times again;

[0063] (3) The chloroform solution was concentrated under reduced pressure and chromatographed to obtain the methyl parathion hapten.

[0064] In a preferred embodiment, in step (1), the mass volume ratio of O,O-diethylthiophosphoryl chloride, p-hydroxybenzoic acid, potassium hydroxide and methanol can be 2.26g:1.38g:1.40g:50mL, but is not limited thereto, which is more conducive to further improving the reaction effect.

[0065] In a preferred embodiment, in step (1), the stirring temperature is 0° C. and the stirring time is 12 h.

[0066] In a preferred embodiment, in step (2), the volume-to-mass ratio of distilled water to solute can be 20 mL:2.26 g, but is not limited thereto, which is more conducive to further improving the extraction effect.

[0067] In a preferred embodiment, in step (2), the concentration of hydrochloric acid may be 6 mol / L, but is not limited thereto.

[0068] In a preferred embodiment, in step (3), the chromatography column may be silica gel, and the chromatography fluid may be CHCl3-MeOH (6:1), but is not limited thereto.

[0069] According to a third aspect of the present invention, there is provided a sibutramine hapten, the structural formula of which is shown in formula (II):

[0070]

[0071] The sibutramine hapten of the present invention is named by systematic nomenclature: 1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutylamine.

[0072] The sibutramine hapten provided by the present invention can obtain a novel artificial antigen structure after being coupled with a carrier protein.

[0073] According to a fourth aspect of the present invention, there is provided a method for preparing the above-mentioned sibutramine hapten, comprising the following steps:

[0074] 1-(4-chlorophenyl)cyclobutanecarbonitrile reacts with isobutylmagnesium bromide and sodium borohydride to obtain sibutramine hapten.

[0075] In the present invention, 4-chlorobenzeneacetonitrile and 1,3-dibromopropane react to generate 1-(4-chlorophenyl)cyclobutanecarbonitrile, which reacts with isobutylmagnesium bromide and sodium borohydride to generate sibutramine hapten (1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutylamine), which has the structure shown in formula (II).

[0076] In the present invention, dimethyl sulfoxide containing potassium hydroxide is dripped into an ether solution containing 4-chlorophenylacetonitrile and 1,3-dibromopropane, and washed to obtain a colorless oily substance 1-(4-chlorophenyl)cyclobutanecarbonitrile; magnesium powder, isobutyl bromide and ether are mixed to obtain an isobutylmagnesium bromide solution; 1-(4-chlorophenyl)cyclobutanecarbonitrile is dripped into the isobutylmagnesium bromide solution, the ether is evaporated, and then an isopropanol suspension containing sodium borohydride is added, the solvent is evaporated, and then extraction is performed with ethyl acetate, and drying is performed over anhydrous magnesium sulfate to obtain sibutramine hapten.

[0077] The preparation method of sibutramine hapten provided by the present invention has simple process and high success rate.

[0078] A typical preparation method of sibutramine hapten, schematic diagram is shown in Figure 2 , including the following steps:

[0079] (1) Pour 4-chlorobenzeneacetonitrile, 1,3-dibromopropane and diethyl ether into a constant pressure funnel and add dimethyl sulfoxide solution containing potassium hydroxide dropwise while stirring. After the addition is complete, add ice water and diethyl ether, filter, and wash the filter residue with diethyl ether;

[0080] (2) Pour the washing liquid and the filtrate into a separatory funnel, discard the aqueous layer, extract the oil layer with ether, wash with water, dry with anhydrous magnesium sulfate, evaporate the ether, and then distill under reduced pressure to obtain 1-(4-chlorophenyl)cyclobutanecarbonitrile;

[0081] (3) Add magnesium powder to a three-necked flask, and add anhydrous ether and isobutyl bromide dropwise while stirring to obtain an isobutyl magnesium bromide solution;

[0082] (4) 1-(4-chlorophenyl)cyclobutanecarbonitrile was dissolved in toluene and then added dropwise to an isobutylmagnesium bromide solution while distilling off the ether. The mixture was stirred and then added with a hot sodium borohydride-isopropanol mixture. The mixture was heated to reflux and then allowed to stand at room temperature. The solvent was evaporated to dryness using a rotary evaporator.

[0083] (5) The solute obtained by evaporation is mixed with water and allowed to stand, extracted with ethyl acetate, the ester layer is washed with water and then dried over anhydrous magnesium sulfate, and the solvent is evaporated to obtain sibutramine hapten.

[0084] In a preferred embodiment, in step (1), the mass volume ratio of 4-chlorobenzeneacetonitrile, 1,3-dibromopropane and diethyl ether can be 15.15g:21.2g:50mL, but is not limited thereto, which is more conducive to further improving the reaction effect.

[0085] In a preferred embodiment, in step (1), the stirring temperature can be 20°C-25°C, nitrogen needs to be introduced during stirring, and the stirring time can be during the dropwise addition and 30 minutes after the dropwise addition, which is more conducive to further improving the reaction effect.

[0086] In a preferred embodiment, in step (1), the mass volume ratio of potassium hydroxide to dimethyl sulfoxide can be 9.6 g:30 mL, but is not limited thereto.

[0087] In a preferred embodiment, in step (1), the volume ratio of ice water to diethyl ether can be 20 mL:100 mL, but is not limited thereto.

[0088] In a preferred embodiment, in step (1), the filtered material may be diatomaceous earth.

[0089] In a preferred embodiment, in step (2), the volume ratio of ether to water can be 100 mL:100 mL, but is not limited thereto.

[0090] In a preferred embodiment, in step (2), the extraction with diethyl ether can be performed twice, but is not limited thereto.

[0091] In a preferred embodiment, in step (2), the number of washing with water can be 3 times, but is not limited thereto.

[0092] In a preferred embodiment, in step (2), the temperature condition of the reduced pressure distillation can be 168° C.-169° C., and the pressure condition can be 2666 Pa.

[0093] In a preferred embodiment, in step (3), the mass volume ratio of magnesium powder, anhydrous ether and isobutyl bromide can be 4.8g:80mL:31.5g, but is not limited thereto, which is more conducive to the full generation of isobutyl magnesium bromide.

[0094] In a preferred embodiment, in step (4), the mass volume ratio of 1-(4-chlorophenyl)cyclobutanecarbonitrile and toluene can be 25 g:100 mL, but is not limited thereto.

[0095] In a preferred embodiment, in step (4), the mass volume ratio of sodium borohydride to isopropyl alcohol can be 15 g:500 mL, but is not limited thereto.

[0096] In a preferred embodiment, in step (4), the heating reflux time can be 6 hours, but not limited thereto; the room temperature storage time can be 18 hours, but not limited thereto.

[0097] In a preferred embodiment, in step (5), the volume ratio of water, ethyl acetate and washing water can be 500 mL:100 mL:100 mL, but is not limited thereto; and the standing time can be 30 min, but is not limited thereto.

[0098] In a preferred embodiment, in step (5), the number of extractions with ethyl acetate can be 3 times, but not limited thereto; the number of washings with water can be 2 times, but not limited thereto.

[0099] According to a fifth aspect of the present invention, a methyl parathion-sibutramine artificial antigen is provided, which is obtained by coupling the above-mentioned methyl parathion hapten and the above-mentioned sibutramine hapten with a carrier protein through the active ester method.

[0100] The methyl parathion-sibutramine artificial antigen provided by the present invention is a novel artificial antigen structure. The polyclonal antibody obtained from the artificial antigen can simultaneously recognize methyl parathion and sibutramine with good recognition ability.

[0101] In a preferred embodiment, the carrier protein may be bovine serum albumin (BSA) and chicken ovalbumin (OVA).

[0102] The first preparation method is to first couple the methyl parathion hapten with BSA / OVA, and then couple it with the sibutramine hapten to construct the graph of different hapten concentrations and inhibition rates, see Figure 3 , calculate the optimal concentration ratio and inhibition rate. The second preparation method: first couple sibutramine hapten with BSA / OVA, then couple it with methyl parathion hapten to obtain the image, see Figure 4 , calculating the optimal concentration ratio and inhibition rate. Comparing the two inhibition rates, the final result showed that the inhibition rate was better when the methyl parathion hapten was first combined with the methyl parathion hapten at a concentration ratio of 8.65:1 (methyl parathion: sibutramine). Therefore, the first preparation method was selected to prepare the methyl parathion-sibutramine artificial antigen.

[0103] By coupling the two haptens with OVA / BSA successively, the effects of the binding order and the concentration ratio of the two drugs on the inhibition rate were explored, thereby obtaining the optimal coupling method; the haptens were coupled to the carrier protein according to methyl parathion-BSA / OVA-sibutramine to obtain artificial antigens.

[0104] It can be seen that the present invention provides a new idea for preparing the best spectrum antibody by exploring the influence of the binding sequence and concentration ratio of two haptens on the inhibition rate.

[0105] A typical preparation method of a methyl parathion-sibutramine artificial antigen comprises the following steps:

[0106] First, the methyl parathion hapten represented by formula (I) is coupled to the carrier protein by the active ester method, and then the obtained conjugate is coupled to the sibutramine hapten represented by formula (II) by the active ester method. After dialysis, the methyl parathion-sibutramine artificial antigen is obtained.

[0107] According to a sixth aspect of the present invention, there is provided a methylparathion-sibutramine polyclonal antibody obtained by immunizing an animal with any of the artificial antigens described above.

[0108] The methyl parathion-sibutramine polyclonal antibody provided by the present invention can simultaneously identify both methyl parathion and sibutramine drugs, has good specificity and high sensitivity, provides core raw materials for establishing a specific immunoassay method for methyl parathion and sibutramine, and has broad development prospects.

[0109] Methylparathion-sibutramine artificial antigen was emulsified and then used to immunize rabbits. The rabbit serum was collected and separated and purified by salting-out method to obtain methylparathion-sibutramine polyclonal antibody.

[0110] In a preferred embodiment, the salting-out method may be an octanoic acid-saturated ammonium sulfate salting-out method.

[0111] A typical preparation method of a methyl parathion-sibutramine polyclonal antibody comprises the following steps:

[0112] First, the methyl parathion-sibutramine artificial antigen was emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and then rabbits were immunized. Then, an equal volume of the methyl parathion-sibutramine artificial antigen was emulsified with Freund's incomplete adjuvant. The rabbits were boosted once every three weeks for a total of three booster immunizations. One week after the third booster immunization, rabbit serum was collected and the antibodies were purified using the octanoic acid-saturated ammonium sulfate method to obtain methyl parathion-sibutramine polyclonal antibodies.

[0113] The polyclonal antibody provided by the present invention has a half inhibitory concentration (IC 50 ) was 4.59 ng / mL, the quantitative detection range was 2.89 ng / mL-7.28 ng / mL, and the detection limit was 2.21 ng / mL; the half inhibitory concentration (IC 50 ) was 59.76 ng / mL, the quantitative detection range was 37.65 ng / mL-94.87 ng / mL, and the detection limit was 28.73 ng / mL.

[0114] According to a seventh aspect of the present invention, there is provided a use of the above-mentioned methyl parathion-sibutramine polyclonal antibody in immunoassay of methyl parathion and sibutramine.

[0115] The application of the methyl parathion-sibutramine polyclonal antibody provided by the present invention solves the technical problem that there is a lack of a highly sensitive and rapid method for simultaneously detecting methyl parathion and sibutramine in the prior art.

[0116] According to an eighth aspect of the present invention, a detection kit for methyl parathion and sibutramine is provided, wherein the detection kit comprises the methyl parathion-sibutramine polyclonal antibody described above.

[0117] The detection kit for methyl parathion and sibutramine provided by the present invention can simultaneously identify the two drugs methyl parathion and sibutramine, and has good specificity and high sensitivity.

[0118] According to a ninth aspect of the present invention, there is provided a use of the above-mentioned detection kit in immunoassay of methyl parathion and sibutramine.

[0119] The application of the detection kit for methyl parathion and sibutramine provided by the present invention solves the technical problem of the lack of a highly sensitive and rapid method for simultaneously detecting methyl parathion and sibutramine in the prior art.

[0120] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0121] Example 1

[0122] A method for preparing methyl parathion hapten comprises the following steps:

[0123] (1) 2.26 g of O,O-diethylphosphorylthiochlorid was added dropwise to a mixture containing 1.38 g of p-hydroxybenzoic acid, 1.4 g of potassium hydroxide, and 50 mL of methanol. The mixture was stirred at 0°C for 12 h, filtered, and the solvent was removed under reduced pressure.

[0124] (2) Add 20 mL of distilled water, extract with chloroform several times, acidify with 6 mol / L hydrochloric acid, and then extract with chloroform several times;

[0125] (3) The chloroform solution was concentrated under reduced pressure and purified by column chromatography (silica gel, CHCl3-MeOH (6:1)) to obtain methyl parathion hapten.

[0126] Example 2

[0127] A method for preparing a sibutramine hapten comprises the following steps:

[0128] Preparation of 1-(4-chlorophenyl)cyclobutanecarbonitrile:

[0129] (1) Pour 9.6 g of potassium hydroxide and 30 mL of dimethyl sulfoxide into a 100 mL three-necked flask, dissolve 15.15 g of 4-chlorobenzeneacetonitrile and 21.2 g of 1,3-dibromopropane in 50 mL of ether, and pour into a 100 mL constant pressure funnel. Stir thoroughly at 20°C-25°C.

[0130] (2) The solution in the constant pressure funnel was slowly dripped into a three-necked flask under nitrogen protection. Stirring was continued for 30 min after the dripping, followed by the dropwise addition of 20 mL of ice water and 100 mL of ether.

[0131] (3) The mixture was filtered through celite, and the residue was washed with ether. The filtrate and washings were combined into a separatory funnel, and the aqueous layer was discarded. The oil layer was extracted twice with 100 mL of ether. The combined ether layers were washed three times with 100 mL of water and dried over anhydrous magnesium sulfate.

[0132] (4) Evaporate the ether from the mixture, and distill the residual orange oil under reduced pressure (168°C-169°C, 2666 Pa) to obtain colorless 1-(4-chlorophenyl)cyclobutanecarbonitrile;

[0133] Preparation of isobutylmagnesium bromide:

[0134] Add 4.8 g of magnesium powder to a three-necked flask, add 80 mL of anhydrous ether dropwise while stirring, and then slowly add 31.5 g of isobutyl bromide to obtain an isobutyl magnesium bromide solution;

[0135] Preparation of 1-[1-(4-chlorophenyl)cyclobutyl]-3-methylbutylamine:

[0136] (1) Dissolve 25 g of 1-(4-chlorophenyl)cyclobutanecarbonitrile in 100 mL of dry toluene, then add dropwise to the isobutylmagnesium bromide solution while distilling off the ether. Adjust the addition rate so that all the ether is evaporated when the addition is complete.

[0137] (2) The mixture was stirred at 90°C for 18 h and then added to 500 mL of isopropanol containing 15 g of hot sodium borohydride. The mixture was heated under reflux for 6 h and allowed to stand at room temperature for 18 h before evaporating the solvent using a rotary evaporator.

[0138] (3) Add 500 mL of water to the residue and mix well. Let it stand for 30 min, extract it three times with 100 mL of ethyl acetate, wash the ester layer twice with 100 mL of water, dry it over anhydrous magnesium sulfate, and evaporate the solvent to obtain a light yellow sibutramine hapten.

[0139] Example 3

[0140] A methyl parathion-sibutramine artificial antigen and its synthesis method and identification:

[0141] Synthesis of methyl parathion-sibutramine artificial antigen:

[0142] (1) Weigh 4.5 mg of methyl parathion hapten, 2 mg of NHS, and 3 mg of EDC and dissolve them in 100 μL of DMF and stir overnight;

[0143] (2) Weigh 9.4 mg of bovine serum albumin (BSA) and add it to 1 mL of PBS buffer;

[0144] (3) Slowly add the solution obtained in step (1) dropwise to the solution obtained in step (2) and stir for 8 h;

[0145] (4) dialyzing with PBS buffer for two days, four times a day, to obtain methyl parathion-BSA artificial antigen after the dialysis is completed;

[0146] (5) Weigh 11.2 mg of sibutramine hapten, 2 mg of NHS, and 3 mg of EDC, dissolve in 100 μL of DMF, and stir overnight;

[0147] (6) Slowly add the solution obtained in step (5) dropwise to the solution obtained in step (4) and stir for 8 h;

[0148] (7) Dialysis with PBS buffer for two days, four times a day, to obtain the methyl parathion-BSA-sibutramine artificial antigen after the dialysis is completed;

[0149] Formula of phosphate buffer solution: Na2HPO4·12H2O 2.90g, NaCl 8.50g, KCl 0.20g, KH2PO4 0.20g, distilled water to 1000mL;

[0150] Similarly, chicken ovalbumin (OVA) was used to replace BSA as a carrier protein to obtain the target product, methyl parathion-OVA-sibutramine artificial antigen. The preparation process was the same as that of the preparation of methyl parathion-BSA-sibutramine artificial antigen.

[0151] Identification of the Methylparathion-Sibutramine Artificial Antigen:

[0152] Take methyl parathion-sibutramine artificial antigen and perform UV full wavelength scanning, such as Figure 5 shown.

[0153] BSA, OVA, and artificial antigens were identified by UV scanning (200nm-400nm), and the maximum absorbance values ​​of each substance before and after coupling were compared. The absorption curve of the methyl parathion-sibutramine artificial antigen was significantly different from that of the carrier protein, indicating that the haptens of methyl parathion and sibutramine were successfully coupled to the carrier protein to produce artificial antigens.

[0154] Example 4

[0155] A method for preparing a methyl parathion-sibutramine polyclonal antibody comprises the following steps:

[0156] (1) New Zealand white rabbits were immunized with the immunogen methyl parathion-BSA-sibutramine. For the first immunization, the immunogen was emulsified with Freund's complete adjuvant at a volume ratio of 1:1, and the rabbits were immunized. Subsequently, the immunogen was emulsified with Freund's incomplete adjuvant at a volume ratio of 1:1, and booster immunization was performed every three weeks for a total of three booster immunizations.

[0157] (2) One week after the third booster immunization, rabbit serum was collected and centrifuged at 4°C and 12,000 rpm for 15 min to remove the precipitate;

[0158] (3) 1 volume of the supernatant was mixed with 2 volumes of acetate buffer, the pH was adjusted to 4.8, 75 μL of octanoic acid was added dropwise while stirring, and the mixture was stirred at room temperature for 30 min, and then allowed to stand at 4°C for 2 h;

[0159] (4) Centrifuge at 4°C and 12,000 rpm for 15 min, filter the supernatant, add 1 / 10 volume of PBS buffer (0.1 M, pH 7.4) to the supernatant, adjust the pH to 7.4 with 2 M sodium hydroxide, and calculate the total volume of the solution;

[0160] (5) Add 0.28 g / mL ammonium sulfate within 30 minutes under ice bath to make a 45% saturated solution, let it stand at 4°C for 1 hour, centrifuge at 4°C and 12,000 rpm for 15 minutes, discard the supernatant, and dialyze the precipitate with PBS buffer (0.1 M, pH 7.4) for three days to obtain methyl parathion-sibutramine polyclonal antibody.

[0161] Example 5

[0162] ELISA test of methyl parathion-sibutramine polyclonal antibodies and its results:

[0163] ELISA test:

[0164] (1) Dilute the original methyl parathion-OVA-sibutramine with coating solution to 0.125 ng / mL, coat the ELISA plate, add 100 μL per well, incubate at 37°C overnight, discard the coating solution, and wash twice;

[0165] (2) Add 120 μL of blocking solution (5% skim milk powder) to each well and block at 37°C for 30 min. Discard the blocking solution, clap the plate, and dry it at 37°C for later use.

[0166] (3) Dilute the antibody serum 16K-fold with PBST, and dilute the methyl parathion standard and sibutramine standard from 1 μg / mL to 0.001 ng / mL with PBST respectively;

[0167] (4) Add 50 μL of methyl parathion dilution solution to each row of columns 1-3 (three parallel groups), add 50 μL of sibutramine dilution solution to each row of columns 4-6 (three parallel groups), then add 50 μL of antibody dilution solution to each well, incubate at 37°C for 40 min, and wash 5 times;

[0168] (5) Add goat anti-rabbit enzyme-linked secondary antibody (5000-fold dilution), incubate at 37°C for 30 min, wash five times, and clap;

[0169] (6) Add color developing solution and develop color for 10 minutes;

[0170] (7) Add 50 μL of 10% H2SO4 to terminate the reaction and read the OD value at 450 nm;

[0171] Results: The standard curve of methyl parathion-sibutramine polyclonal antibody indirect competition ELISA is as follows: Figure 6 and Figure 7 As shown;

[0172] The half inhibitory concentration (IC50) of methyl parathion-sibutramine polyclonal antibody against methyl parathion 50 ) was 4.59 ng / mL, and the linear range of quantitative detection (IC 20 -IC 80 ) was 2.89ng / mL-7.28ng / mL, and the minimum detection limit was 2.21ng / mL; the half-inhibitory concentration (IC 50 ) was 59.76 ng / mL, and the linear range of quantitative detection (IC 20 -IC 80 ) was 37.65ng / mL-94.87ng / mL, and the minimum detection limit was 28.73ng / mL.

[0173] It can be seen that the methyl parathion-sibutramine polyclonal antibody of the present invention can meet the detection requirements and has good recognition ability for both methyl parathion and sibutramine.

[0174] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A methyl parathion-sibutramine artificial antigen, characterized in that: It is obtained by coupling methyl parathion hapten and sibutramine hapten through carrier protein; The structural formula of the methyl parathion hapten is shown in formula (I): Formula (I); The preparation method of the methyl parathion hapten comprises the following steps: O,O-diethylthiophosphoryl chloride reacts with p-hydroxybenzoic acid to obtain the methyl parathion hapten; The structural formula of the sibutramine hapten is shown in formula (II): Formula (II); The preparation method of the sibutramine hapten comprises the following steps: 1-(4-chlorophenyl)cyclobutanecarbonitrile reacts under the action of isobutylmagnesium bromide and sodium borohydride to obtain the sibutramine hapten.

2. The artificial antigen according to claim 1, characterized in that The carrier proteins include bovine serum albumin and chicken ovalbumin.

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