CN112174902A

CN112174902A - Oxazepam hapten, oxazepam antigen, and preparation methods and applications thereof

Info

Publication number: CN112174902A
Application number: CN202011051890.0A
Authority: CN
Inventors: 袁强; 洪裕好; 黄俊兴
Original assignee: Guangzhou Wondfo Biotech Co Ltd
Current assignee: Guangzhou Wondfo Biotech Co Ltd
Priority date: 2020-09-29
Filing date: 2020-09-29
Publication date: 2021-01-05

Abstract

The invention relates to an oxazepam hapten, an oxazepam antigen, a preparation method and an application thereof, wherein the structure of the oxazepam hapten is shown as a formula (I), and the preparation method of the oxazepam hapten comprises the following steps: taking oxazepam and dianhydride as raw materials, introducing carboxyl on a 5-position benzene ring of oxazepam by adopting Friedel-crafts acylation reaction, and obtaining the oxazepam hapten. The preparation method of the oxazepam antigen comprises the following steps: activating an oxazepam hapten, and then carrying out coupling reaction with a carrier protein to obtain the oxazepam antigen. The invention firstly creates an oxazepam artificial hapten and an oxazepam artificial antigen preparation method, has concise and effective synthesis steps, can be completely used in immunoassay, and provides a convenient way for people to detect and research oxazepam in the future.

Description

Oxazepam hapten, oxazepam antigen, and preparation methods and applications thereof

Technical Field

The invention belongs to the technical field of biology, and relates to an oxazepam hapten, an oxazepam antigen, and preparation methods and applications thereof.

Background

Oxazepam (Oxazepam) is a benzodiazepine sedative hypnotic, another name: shuning, chemical name is: 7-chloro-2, 3-dihydro-3-hydroxy-5-phenyl-1, 4(2H) -benzodiazepin-2-one. Oxazepam is a main active metabolite of diazepam, and belongs to short-acting benzodiazepine drugs. In clinic, the oxazepam is used for short-term treatment of anxiety disorder and insomnia related to the anxiety disorder, has little influence on liver function, and is suitable for old people and patients with liver diseases. Many patients take oxazepam tablets for a long time to generate dependence, and withdrawal reaction can occur after the patients stop taking the oxazepam tablets. For this reason many countries place such drugs on the forbidden drug list, strictly regulated. At present, the detection of oxazepam at home and abroad mainly comprises High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), gas-mass combined method (GC-MS), liquid-mass combined method (LC-MS) and the like.

CN107271599A discloses an on-line SPE-LC-MS/MS analysis method of diazepam and metabolites thereof in human saliva, wherein a saliva sample is subjected to on-line solid phase extraction, and a C18 chromatographic column is adopted for chromatographic separation, and is subjected to gradient elution by mobile phase water (containing 2mmol/L ammonium formate and 0.1% formic acid) and mobile phase acetonitrile (containing 2mmol/L ammonium formate and 0.1% formic acid); mass spectrometry uses electrospray ionization (ESI), Multiple Reaction Monitoring (MRM) mode to detect the target compound. The invention establishes a simple, sensitive, stable and full-automatic online SPE-LC-MS/MS method for quantifying diazepam and 5 metabolites thereof (desmetazem, oxazepam, temazepam, oxazepam glucuronide conjugate and temazepam glucuronide conjugate) in human saliva for the first time.

CN109085265A discloses a kit for detecting anxiolytic/hypnotic drugs in serum plasma by liquid chromatography tandem mass spectrometry, which comprises: the pharmaceutical standard substance is as follows: bromazepam, clonazepam, diazepam, lorazepam, midazolam, nitrazepam, oxazepam, temazepam; pharmaceutical internal standard compound: alprazolam-d 5, clonazepam-d 4, diazepam-d 5, lorazepam-d 4, midazolam-d 4, nitrazepam-d 5, oxazepam-d 5, temazepam-d 5; the medicine extraction composition comprises the following components: 60% methanol solution, 20% acetonitrile solution, 10% isopropanol solution and 10% purified water by volume; negative plasma; and a diluent: 50% methanol aqueous solution, the kit can be used for simultaneously detecting the anxiolytic/hypnotic drugs and the active metabolites thereof, and has short detection time and large flux.

However, these methods not only require expensive instruments and equipment, but also have high requirements for materials to be tested, and require further purification treatment, which has not yet met the requirements of modern detection on rapidness, convenience and accuracy. In order to strengthen the supervision of the medicine and guarantee the physical health of people, research on benzodiazepine immunoassay methods needs to be carried out, and an effective preparation method of the oxazepam artificial antigen needs to be provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an oxazepam hapten, an oxazepam antigen, a preparation method and an application thereof, and specifically comprises the oxazepam hapten, the preparation method of the oxazepam hapten, the oxazepam antigen, the preparation method of the oxazepam antigen, the application of the oxazepam hapten or the oxazepam antigen in immunodetection of benzodiazepines compounds in a sample, and an immunochromatographic kit for detecting the benzodiazepine compounds in the sample.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides an oxazepam hapten, wherein the structure of the oxazepam hapten is shown as a formula (I):

wherein x is any integer from 2 to 6, for example, x is 2,3, 4, 5 or 6; y is 2 times x.

The invention provides a novel oxazepam artificial antigen which is applicable to immunoassay of benzodiazepine compounds, particularly oxazepam, and improves the speed, convenience and accuracy of detection of the benzodiazepine compounds. The antigen prepared by the invention can reduce the cross-product interference of a detection kit and improve the detection rate of the benzodiazepine drugs, can be applied to the kit of hair detection materials, improves the detection sensitivity to 3ng/ml, and is the highest in sensitivity among similar products.

In a second aspect, the invention provides a method of preparation of an oxazepam hapten as described above, the method of preparation of the oxazepam hapten comprising: taking oxazepam and dianhydride as raw materials, introducing carboxyl on a 5-position benzene ring of oxazepam by adopting Friedel-crafts acylation reaction, and obtaining oxazepam hapten; the dianhydride includes succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride or suberic anhydride.

The key factor of the synthesis method of the oxazepam hapten related by the invention is that carboxyl is introduced into an oxazepam molecular structure, the oxazepam hapten is screened by a plurality of synthesis schemes, a Friedel-crafts acylation reaction is adopted, the carboxylation process condition is mild, high temperature and high pressure are not needed, the operation is simple, and the reaction yield is high.

Preferably, a catalyst is used in the reaction.

Preferably, the molar ratio of the dianhydride to the catalyst is 1 (1-3), such as 1:1, 1:1.5, 1:2, 1:2.5 or 1:3, and other specific values within the above range can be selected, and are not described in detail herein.

The molar ratio of dianhydride to catalyst is specifically selected to be within the numerical range of 1 (1-3) because if the catalyst is present in a ratio below this range, the acylation will be incomplete and the reactants will not react completely resulting in waste; if the proportion of the catalyst is higher than the range, both the acyl and the acid radical ions are complexed with the catalyst.

Preferably, the catalyst comprises aluminium chloride.

Aluminum chloride is preferred as a catalyst for the synthesis reaction in the present invention because aluminum chloride has higher catalytic activity and is easily available compared to other Lewis acids. Catalytic activity of Lewis acid catalyst: AlCl₃＞FeCl₃＞SbCl₃＞BF₃＞TiCl₄＞ZnCl₂。

Preferably, the molar ratio of oxazepam to dianhydride is 1:2-2:1, for example, 1:2, 2:3, 1:1, 3:2 or 2:1, and other specific values within the above range can be selected, and are not described in detail herein.

The molar ratio of oxazepam and dianhydride is selected to be 1:2-2:1, because if the addition amount of dianhydride is lower than the range, a large amount of main reactant oxazepam is not involved in the reaction and is wasted, and if the addition amount of dianhydride is higher than the range, acyl and acid radical ions generated in the Friedel-crafts reaction are complexed with aluminum trichloride.

Preferably, the reaction is carried out in an anhydrous medium.

Preferably, the anhydrous medium comprises any one or a combination of at least two of dichloromethane, chloroform, carbon tetrachloride, diethyl ether or acetone; the combination of at least two of the above-mentioned compounds, such as the combination of dichloromethane and chloroform, the combination of carbon tetrachloride and ether, the combination of ether and acetone, etc., other arbitrary combination modes can be selected, and are not repeated herein. Dichloromethane is preferred.

The reason why dichloromethane is preferred as a reaction medium in the above synthesis reaction is that dichloromethane is not mixed into water in the air at low temperature; after the reaction is over, dichloromethane is more easily removed and toxicity is low.

Preferably, the reaction temperature is 15-35 ℃, for example, 15 ℃, 20 ℃, 22 ℃, 25 ℃, 30 ℃ or 35 ℃, and the time is 10-24h, for example, 10h, 12h, 15h, 18h, 20h or 24h, and other specific values within the above range can be selected, and are not repeated herein.

When the preparation method of the oxazepam hapten related by the invention meets the requirements of temperature, time, raw material proportion, catalyst and medium, the reaction efficiency is higher, and the yield of the oxazepam hapten is higher.

In a third aspect, the invention provides an oxazepam antigen prepared from an oxazepam hapten as described in the first aspect.

The oxazepam hapten related to the invention is activated by coupling with a carrier protein, and the modified antigen with the carrier protein has stronger immune activity, because a single small molecule does not have immune activity or has low immune activity.

In a fourth aspect, the invention provides a method of preparation of an oxazepam antigen as described above, the method of preparation comprising: activating an oxazepam hapten, and then carrying out coupling reaction with a carrier protein to obtain the oxazepam antigen.

Preferably, the oxazepam hapten is activated in a manner that: mixing the oxazepam hapten with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for reaction to obtain the activated oxazepam hapten.

Preferably, the molar ratio of the oxazepam hapten, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 (1-1.5) to (1-1.5), such as 1:1:1, 1:1:1.5, 1:1.5:1, 1:1.5:1.5, 1:1.2:1, 1:1:1.2 or 1:1.2:1.2, and the like, and other specific point values in the ranges can be selected, so that the description is omitted.

Preferably, the reaction medium of the reaction comprises N, N-dimethylformamide.

Preferably, the carrier protein comprises bovine serum albumin, ovalbumin, hemocyanin, bovine hemoglobin, bovine thyroid protein, human immunoglobulin IgG or human immunoglobulin IgA.

Preferably, the pH of the coupling reaction system is maintained at 9.4-9.8, for example, pH 9.4, pH 9.5, pH9.6, pH 9.7, or pH 9.8, and other specific values within the above range can be selected, which is not described in detail herein.

Preferably, the temperature of the coupling reaction is 2-6 ℃, for example, 2 ℃,3 ℃,4 ℃, 5 ℃ or 6 ℃, and the time is 10-24h, for example, 10h, 12h, 15h, 18h, 20h or 24h, and other specific values within the above range can be selected, and are not repeated herein.

Preferably, after the coupling reaction is finished, the reaction mixture is purified by dialysis, and the dialysis solution is 0.05M PBS buffer solution with pH 7.4.

When the preparation method of the oxazepam antigen meets the requirements of temperature, time, raw material proportion, system pH environment and medium, the reaction efficiency is higher, and the yield of the oxazepam antigen is higher.

In a fifth aspect, the invention provides an oxazepam hapten as described above or an oxazepam antigen as described above for use in the immunoassay of a benzodiazepine compound, particularly oxazepam, in a sample.

Preferably, the sample comprises a body fluid sample or a tissue sample.

The oxazepam hapten or the oxazepam antigen synthesized by the invention can be used for detecting whether a sample contains benzodiazepines substances or not by utilizing an immunoreaction. The sample herein refers to a sample of a mammalian or human body fluid, such as saliva, urine, sweat; alternatively, a tissue sample from a mammal or human, such as hair, liver, spleen, etc. The "immunoassay" as used herein is an immunoassay or a detection method using the principle of binding an antibody to an antigen, and includes an enzyme-linked immunosorbent assay (ELISA) and also a Lateral Flow test strip (lareal Flow) method, including a competition method.

The lateral flow test strips are made of absorbent or non-absorbent materials, and one test strip may be made of a variety of materials for fluid transfer. One material of the test strip may be superimposed on another test strip material, such as filter paper superimposed on nitrocellulose. Alternatively, one region of the test strip containing at least one material is positioned behind another region containing at least one different material. In this case, the liquid can flow between the regions, and they may or may not overlap each other. The material on the test strip may be fixed to a support such as a plastic backing or a hard surface to enhance the test strip holding force. The test strip regions may be arranged as follows: a sample addition zone, at least one reagent zone, at least one detection result zone, at least one control zone, at least one adulteration detection zone and a liquid absorption zone. If the detection zone comprises a control zone, it is preferred that the control zone is located after the analyte detection zone in the detection result zone. All of these regions or combinations thereof may be on a single test strip containing one material. In addition, the zones are made of different materials and are joined together in the direction of liquid transfer. For example, the different zones may be in direct or indirect fluid communication.

In a sixth aspect, the invention provides an immunochromatographic kit for detecting benzodiazepine compounds in a sample, which comprises a sample pad, a marking pad, a nitrocellulose membrane, a water absorption plate and a bottom plate; the nitrocellulose membrane comprises a detection line coated by the oxazepam antigen and a quality control line coated by a goat anti-mouse antibody.

The preparation method of the sample pad comprises the following steps: and soaking the glass fiber in the sample solution, and drying.

The preparation method of the marking pad comprises the following steps: (1) HAuCl₄The aqueous solution and the aqueous solution of trisodium citrate are azeotroped to prepare the colloidal gold solution, and the color is red. (2) Combining colloidal gold with protein (IgG), adding an oxazepam antibody solution into a colloidal gold solution with the pH of 9.0, adding a stabilizer, and performing centrifugal purification to obtain the immune colloidal gold marker. (3) And coating the prepared immune colloidal gold marker solution with the concentration on glass fiber, and drying to obtain the marker pad.

The preparation method of the nitrocellulose membrane comprises the following steps: a T line solution is prepared from an oxazepam-protein conjugate (an oxazepam antigen related to the invention), a C line solution is prepared from a goat anti-mouse antibody, and then T line (detection line) and C line (quality control line) areas on a nitrocellulose membrane are respectively coated.

The preparation method of the kit comprises the following steps: the sample pad, the marking pad, the nitrocellulose membrane coated with the T line and the C line and the water absorption plate are arranged in sequence from the sample adding end. The four parts are all adhered on the bottom plate in sequence and then cut into required width to form the reagent strip. Or can be filled according to the size of a mould (plastic shell) to form the cassette type kit.

Compared with the prior art, the invention has the following beneficial effects:

the invention creates a novel oxazepam artificial hapten and an oxazepam artificial antigen for the first time, the preparation method of the invention has simple and effective synthetic steps, can be completely used in immunoassay, provides a convenient way for the research of oxazepam by people in the future, and can meet the domestic needs of the research.

Drawings

FIG. 1 is an ESI-MS analysis spectrum of oxazepam hapten prepared in example 1;

FIG. 2 is a UV scan of oxazepam hapten, bovine serum albumin and oxazepam antigen from examples 1, 3;

FIG. 3 is an external view of the immunochromatographic diagnostic kit for detecting benzodiazepines compounds prepared in example 4;

FIG. 4 is a schematic view showing the results of the immunochromatographic diagnostic kit prepared in example 4 in detecting benzodiazepines (a is a negative result, b is a positive result, and c and d are both failure results).

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

In this example, an oxazepam hapten is prepared by the following method:

respectively weighing 10mmol of anhydrous aluminum trichloride and 5mmol of succinic anhydride in a 50mL round-bottom flask, adding 10mL of dichloromethane, and stirring at 0 ℃; weighing 5mmol of oxazepam, adding into the reaction solution for 3 times, heating to 25 ℃, and reacting for more than 12 hours; slowly adding 20mL of ultrapure water under vigorous stirring, standing for layering, taking an organic layer, carrying out reduced pressure distillation, dissolving the obtained residue with 5mL of ethyl acetate, washing with saturated sodium chloride solution twice, 5mL each time, drying and evaporating the organic layer, and purifying by column chromatography to obtain 1.8mmol of hapten for later use. ESI-MS analysis (385.1[ M-1]) was performed on the obtained hapten, and the result is shown in FIG. 1, which confirmed that oxazepam hapten was successfully obtained.

Monitoring the reaction end point: monitoring was carried out by thin layer chromatography using coating thickness: 0.2 +/-0.03 mm, silica gel powder granularity: (HPTLC): more than or equal to 80 percent of 8 +/-2 um, (TLC): 10-40 μm, scattering parameter: SX ═ 3, pH: 6.2-6.8, specification: a 2.5 multiplied by 7.5cm thin layer chromatography silica gel plate, wherein 1cm position at the lower end of the silica gel plate is taken as an initial line, a 0.3mm sample application capillary is sample applied on the line, the thin layer plate is placed into a chromatography cylinder after the sample application is finished and dried, the chromatography liquid is ethyl acetate and petroleum ether with the volume ratio of 1:2, the thin layer plate is unfolded to 0.5-0.8cm position at the upper end of the thin plate, the plate is taken out, and a developing agent is dried; the sheet was placed in an ultraviolet analyzer and observed at a wavelength of 254nm, and at an Rf value of 0.53, a color developing point was observed as a reaction end point.

Example 2

In this example, an oxazepam hapten is prepared by the following method:

respectively weighing 20mmol of anhydrous aluminum trichloride and 5mmol of succinic anhydride in a 50mL round-bottom flask, adding 10mL of dichloromethane, and stirring at 0 ℃; weighing 5mmol of oxazepam, adding into the reaction solution for 3 times, heating to 25 ℃, and reacting for more than 12 hours; slowly adding 20mL of ultrapure water under vigorous stirring, standing for layering, then obtaining a large amount of flocculent precipitate, taking an organic layer, carrying out reduced pressure distillation, dissolving the obtained residue with 5mL of ethyl acetate, washing with saturated sodium chloride solution twice, each time with 5mL, drying and evaporating the organic layer, and purifying by column chromatography to obtain 0.03mmol of hapten. The hapten thus obtained was compared with the hapten obtained in example 1 and found to be the same substance, but the amount of hapten obtained was 1.67% of that obtained in example 1.

This example shows too much catalyst, which becomes precipitate by complexing with acyl group of hapten, resulting in low yield of target product.

Example 3

In this example, an oxazepam antigen is prepared by the following method:

(1) preparation of oxazepam hapten active ester:

in a 25mL dry single-neck flask, 1mmol of the hapten prepared in example 1, 1.2mmol of N-hydroxysuccinimide, 1.2mmol of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 10mL of N, N-dimethylformamide are added and stirred under protection of helium at 25 ℃ for 18 h. After the reaction is finished, the reaction mixture is divided into 8 centrifuge tubes with the volume of 1.5mL, the centrifuge tube is centrifuged at 10000rpm for 30min, and the supernatant is divided into 8 glass sealed bottles with the volume of 2mL and stored for standby under the protection of helium.

(2) Preparation of oxazepam antigen (oxazepam-bovine serum albumin):

100mg of BSA was put into a 25mL single-neck flask, 8mL of 0.1M sodium carbonate-sodium bicarbonate buffer (pH9.6) and 1.4mL of N, N-dimethylformamide were added thereto and dissolved under stirring, the temperature of the reaction mixture was maintained at about 4 ℃ by an ice-water bath, 2.6mL of an active ester solution was slowly added dropwise to the BSA solution, and the pH of the reaction mixture was adjusted with 0.1M sodium hydroxide solution as needed to maintain the pH of the mixture at about 9.6. After the dropwise addition, the temperature of the reaction solution was maintained at about 4 ℃ and the reaction was stirred for 18 hours. After the reaction is finished, transferring the reaction mixture into a dialysis bag, dialyzing for multiple times by using 0.05M PBS buffer solution with pH7.4, monitoring by using an ultraviolet spectrophotometer until the light absorption value of the dialysate is consistent with the baseline at 200-400nm, and then freeze-drying the liquid in the dialysis bag to obtain the target antigen: oxazepam-bovine serum albumin.

The prepared oxazepam antigen is identified as follows:

(1) and (3) coupling ratio determination: the binding ratio of the conjugate is the ratio of the number of molecules of hapten to carrier protein in the conjugate, and the binding ratio can be determined by spectrophotometry. Spectrophotometry is a principle that the absorption of light by a substance is in a proportional relationship with the concentration of the substance, and the concentrations of two coupled molecules are respectively measured. In the macromolecular and micromolecular conjugate, the two molecules have different ultraviolet scanning spectrums respectively and show the superposed properties of the spectrums. The binding ratio of the conjugate is the ratio of the number of molecules of hapten to carrier protein in the conjugate.

Respectively measuring the molar extinction coefficients () of the oxazepam hapten, bovine serum albumin and the oxazepam antigen under a certain wavelength, and according to the additive principle of absorbance in spectral analysis and a formula: the number of moles of hapten coupled per mole of carrier protein was calculated. According to the ultraviolet spectrum scanning result, qualitative analysis can be carried out by contrasting the ultraviolet absorption spectrum characteristics of the three.

An ultraviolet scan of the oxazepam hapten, the bovine serum albumin and the oxazepam antigen is shown in figure 2, BSA has typical absorption at 279nm and 252nm, and absorption of the oxazepam hapten and the oxazepam-bovine serum albumin conjugate has obvious and deviated at 279nm and 252nm, which indicates that the artificial antigen conjugate has different ultraviolet absorption characteristics with a precursor substance thereof, indicates that the hapten and the carrier protein are successfully coupled, and the molecular binding ratio of the hapten to the carrier protein is calculated to be 7.05 according to the formula.

(2) Determination of protein concentration of oxazepam antigen (oxazepam-bovine serum albumin): preparing 1.5mL of bovine serum albumin solution with the concentration of 0, 40, 60, 80, 100, 120, 160 and 200 mu g/mL, adding 5mL of Coomassie brilliant blue staining solution, immediately mixing uniformly, standing for 20 minutes, taking parallel samples at each concentration, measuring the light absorption value at 595nm, and drawing a relation curve between the protein concentration and the light absorption value. Diluting the antigen solution according to a certain proportion, measuring the light absorption value of the antigen solution at 595nm, and obtaining the corresponding protein concentration of the antigen solution from the curve. The protein concentration of the antigen solution calculated by the present invention was 8.57 mg/mL.

Example 4

The immunochromatography diagnostic kit for detecting benzodiazepine compounds prepared in the embodiment is prepared by the following steps:

(1) sample pad preparation: soaking glass fiber in a sample pad treatment solution, and then drying, wherein the sample pad treatment solution is 0.05M PBS, and meanwhile, the PBS solution also contains the following raw materials in mass ratio: 0.1% BSA, 0.01% LowCross-Buffer, 0.01% trehalose, 0.1% NaCl, 0.05% PVP-10, 0.02% Triton X-100, 0.02% Tween-80, 0.005% Proclin 300.

(2) Preparing a marking pad: taking HAuCl with the mass fraction of 0.01 percent₄Adding 0.75mL of trisodium citrate aqueous solution with the mass fraction of 1 percent into 100mL of the aqueous solution, and heating and boiling for 30min to turn red. Stopping heating, and cooling for later use. Mixing the colloidal gold solution with 0.1M K₂CO₃Adjusting pH to 9.0, stirring the colloidal gold solution, adding anti-oxazepam monoclonal antibody (provided by a Wanfu organism), continuously stirring for 10min, centrifuging and purifying to obtain an immune colloidal gold marker, coating the immune colloidal gold marker on glass fiber, and drying for later use.

(3) Preparing a nitrocellulose membrane: a T line solution is prepared by using the oxazepam antigen prepared in example 3, a C line solution is prepared by using a goat anti-mouse IgG antibody, and then the T line solution and the C line solution are respectively coated on a T line area and a C line area on a nitrocellulose membrane.

(4) The pasting plate mode is as follows: the nitrocellulose membrane is connected with the marking pad in the direction of a T line (detection line) and is connected with the water absorption plate in the direction of a C line (quality control line). The label pad pressed the nitrocellulose membrane 2 mm. The sample pad is connected with the marking pad, and the lower part of the sample pad is flush with the lower end of the PVC bottom plate at the position of the marking pad 1/3. The nitrocellulose membrane is pressed by the water absorption plate for 1mm, and the upper end of the nitrocellulose membrane is flush with the upper end of the PVC bottom plate. When the detection sample is dripped to the sample pad, the sample is chromatographed upwards by utilizing the capillary effect, and the sample moves along the directions of the sample pad, the marking pad, the nitrocellulose membrane and the water absorption plate, so that the reaction process is completed. Cutting to form reagent strips, and filling into a mold to form a cassette kit, as shown in FIG. 3.

(5) And (3) judging a detection result:

100 mu l of a sample to be detected (urine, saliva or hair decomposition extract) is dripped into a sample adding area of the kit sample, after the sample is dripped on a sample pad, liquid flows to the end of a water absorption plate for chromatography due to capillary chromatography effect, if no oxazepam exists in the sample liquid, colloidal gold particles marked with anti-oxazepam antibodies run to a T line (detection line) position on a membrane along with the sample liquid, and then generate immune binding reaction with oxazepam antigens coated on the T line, and the colloidal gold particles are stacked at the T line position to enable the T line to present a macroscopic red line which is a negative result, as shown in a in figure 4. If the sample liquid contains benzodiazepines, the benzodiazepines compete for limited antibody binding sites with the oxazepam-bovine serum albumin conjugate coated on the T line position, and when the concentration of the benzodiazepines in the sample liquid reaches a certain amount, the benzodiazepines occupy all the antibody binding sites marked on the colloidal gold particles, so that the binding of the oxazepam-bovine serum albumin conjugate coated on the T line position and the antibody colloidal gold thereof is prevented, and thus, a T line area has no red line and shows a positive result, as shown in b in fig. 4. No matter whether the benzodiazepines exist in the sample liquid or not, a red line appears in a C line (quality control line) area, and the red line is used for judging whether enough products to be detected exist or not and whether the chromatography process meets the normal standard or not and is also used as the internal control standard of the kit.

Normal results: the positive is a red line appearing on the line C (quality control line) in the display area of the kit, as shown in a in figure 4; the negativity is that a red line appears on each of a T line (detection line) and a C line (quality control line) in a display area of the kit, as shown in b in figure 4; failure results: in the kit display area, there is no red line on both lines T and C, as shown by d in FIG. 4, or only one red line appears on line T, as shown by C in FIG. 4.

Example 5

In this example, the immunochromatographic diagnostic kit for detecting benzodiazepines compounds prepared in example 4 was used to detect samples derived from clinical samples identified by guangdong fufu medical and toxicology, and the operation method was as follows:

(1) 305 clinical urine samples are randomly taken, and the content of benzodiazepine drugs in the urine samples is quantified through GC/MS, wherein 41 positive samples exist, and 264 negative samples exist. In this example, the interpretation threshold was set at 300ng/ml, above which a positive result (indicated by "+") was obtained and below which a negative result (indicated by "-") was obtained.

(2) And taking the quality control liquid to carry out sensitivity detection on the kit and recording the result.

12 negative quality control products, and the interpretation result of the kit is "-";

3 quality control products with 50% cut off value, and the reading result of the kit is "-";

3 quality control products with 50% cut off value, and the interpretation result of the kit is plus;

the sensitivity meets the product standard.

(3) And (3) taking 100 mu L of a sample to be detected, dripping the sample to be detected in a sample adding area of the kit, and reading and recording the result after 5 min.

The results of the two detection methods were compared:

36 samples with GC/MS quantitative value > 300ng/ml, and the result of the kit interpretation is "+";

5 samples with GC/MS quantitative value of more than 0ng/mg and less than or equal to 300ng/ml are judged to be plus-minus by the kit;

264 samples with GC/MS quantitative value of 0ng/ml are judged to be negative by the kit.

(4) The detection performance of the similar kit is shown in table 1:

TABLE 1 detection Performance of the same type kit

Wherein: "+" is positive and "-" is negative.

As can be seen from Table 1, the kit provided by the invention has a high detection rate for benzodiazepine drugs, and compared with other benzodiazepine detection kits on the market, the kit provided by the invention can also detect a plurality of benzodiazepine drugs such as bromazepam, clobazam, chlordiazepoxide-potassium, dealkylation flurazepam, flurazepam and the like, and the application range of the kit is wide.

(5) In order to embody the anti-interference performance of the kit, the anti-interference test is carried out on 91 medicaments, and the test results are shown in table 2.

TABLE 2 anti-interference Properties of the kit of the invention

Wherein: "+" is positive and "-" is negative.

As can be seen from Table 2, the kit of the technical scheme of the invention has excellent anti-interference performance, shows negative detection results for most of the drugs on the market at present, and has good anti-interference effect.

Example 6

In this example, the immunochromatographic diagnostic kit for detecting benzodiazepines compounds prepared in example 4 was used to detect a sample, which was a quality control product from guangzhou mobilephone biotechnology, ltd, and the operation method was as follows:

(1) and (3) dropwise adding the quality control product of the hair extract of oxazepam with different concentrations into a sample adding area of the kit sample, and reading and recording the result after 5 min.

(2) The results are shown in table 3 below:

TABLE 3 Oxazepam sensitivity assay

Note: "+" indicates positive; "-" indicates negative.

The test result shows that the colloidal gold immunochromatographic kit prepared in example 4 has very high sensitivity, and the lowest detection amount in the hair extract solution can reach 3 ng/mL.

The applicant states that the invention is illustrated by the above examples to describe an oxazepam hapten, an oxazepam antigen, a preparation method and application thereof, but the invention is not limited by the above examples, that is, the invention does not mean that the invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims

1. An oxazepam hapten, which has the structure as shown in formula (I):

wherein x is any integer of 2-6; y is 2 times x.

2. The method of preparation of oxazepam hapten as claimed in claim 1, wherein said method of preparation of oxazepam hapten comprises: taking oxazepam and dianhydride as raw materials, introducing carboxyl on a 5-position benzene ring of oxazepam by adopting Friedel-crafts acylation reaction, and obtaining oxazepam hapten; the dianhydride includes succinic anhydride, glutaric anhydride, adipic anhydride, pimelic anhydride or suberic anhydride.

3. The method for the preparation of oxazepam hapten as claimed in claim 2, wherein a catalyst is used in the reaction;

preferably, the molar ratio of dianhydride to catalyst is 1 (1-3);

preferably, the catalyst comprises aluminum chloride;

preferably, the molar ratio of oxazepam to dianhydride is from 1:2 to 2:1.

4. The method for the preparation of oxazepam haptens according to claim 2 or 3, wherein the reaction is carried out in an anhydrous medium;

preferably, the anhydrous medium comprises any one or a combination of at least two of dichloromethane, chloroform, carbon tetrachloride, diethyl ether or acetone;

preferably, the reaction temperature is 15-35 ℃ and the reaction time is 10-24 h.

5. An oxazepam antigen, wherein said oxazepam antigen is produced from an oxazepam hapten as claimed in claim 1.

6. The method of preparation of an oxazepam antigen of claim 5, wherein the method of preparation comprises: activating an oxazepam hapten, and then carrying out coupling reaction with a carrier protein to obtain the oxazepam antigen.

7. The method of preparation of an oxazepam antigen of claim 6, wherein said oxazepam hapten is activated in a manner that: mixing oxazepam hapten with N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride for reaction to obtain activated oxazepam hapten;

preferably, the molar ratio of the oxazepam hapten, the N-hydroxysuccinimide and the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 (1-1.5) to (1-1.5);

8. The method for producing an oxazepam antigen according to claim 6 or 7, wherein the carrier protein comprises bovine serum albumin, ovalbumin, hemocyanin, bovine hemoglobin, bovine thyroid protein, human immunoglobulin IgG or human immunoglobulin IgA;

preferably, the pH value of the system of the coupling reaction is maintained between 9.4 and 9.8;

preferably, the temperature of the coupling reaction is 2-6 ℃ and the time is 10-24 h;

9. Use of an oxazepam hapten as claimed in claim 1 or an oxazepam antigen as claimed in claim 5 in the immunoassay for a benzodiazepine compound in a sample;

preferably, the sample comprises a body fluid sample or a tissue sample.

10. An immunochromatography kit for detecting benzodiazepine compounds in a sample, which is characterized by comprising a sample pad, a marking pad, a nitrocellulose membrane, a water absorption plate and a bottom plate; the nitrocellulose membrane comprises a detection line coated with the oxazepam antigen of claim 5 and a quality control line coated with a goat anti-mouse antibody.