CN112321443A

CN112321443A - Dextropropoxyphene artificial hapten as well as preparation method and application thereof

Info

Publication number: CN112321443A
Application number: CN202011137918.2A
Authority: CN
Inventors: 袁强; 伍丽贤; 郭锦成
Original assignee: Guangzhou Wondfo Biotech Co Ltd
Current assignee: Guangzhou Wondfo Biotech Co Ltd
Priority date: 2020-10-22
Filing date: 2020-10-22
Publication date: 2021-02-05

Abstract

The invention provides a dextropropoxyphene artificial hapten as well as a preparation method and application thereof, wherein the dextropropoxyphene artificial hapten has a structure shown in a formula I, the synthetic method of the dextropropoxyphene artificial hapten is simple and easy to operate, and the dextropropoxyphene artificial hapten conjugate formed by using the dextropropoxyphene artificial hapten can be used for qualitative or quantitative detection of dextropropoxyphene and can also be applied to an immunochromatography technology.

Description

Dextropropoxyphene artificial hapten as well as preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a dextropropoxyphene artificial hapten as well as a preparation method and application thereof.

Background

Dexpropoxyphene (dexproxyphene) is an opioid analgesic, alias: dafufeng, dextro-Dafufeng and dextro-propoxyphene, the chemical name is: (2S, 3R) -2-benzyl-4- (dimethylamino) -3-methyl-2-phenylbutyric acid ethyl ester. Dextropropoxyphene is an narcotic analgesic used for treating acute and chronic pains, has a chemical structure and a pharmacological action similar to those of methadone, is mainly combined with opioid mu receptors in vivo, but has a weak analgesic effect, is a weak opioid analgesic, is only used for relieving mild to moderate pains, has almost no antitussive effect, and is an opioid medicine which is most widely used for relieving mild to moderate pains; such drugs are also abused by lawbreakers because of the addictive nature of improper use, similar to other opioid analgesics.

Dextromethorphan is listed as an anesthetic in the catalog of anesthetic varieties of the people's republic of China (2013 edition), and is strictly regulated. The 2020 world drug report: the drug market is becoming increasingly complex. In addition to plant substances such as marijuana, cocaine and heroin, hundreds of synthetic drugs have been added, and the non-medical use of drugs is rapidly increasing. Of the total number of new psychoactive species identified in 2014, the opioid new psychoactive species accounted for only 2%, but by 2018 this figure has risen to 9%. At present, the detection of dextropropoxyphene at home and abroad mainly comprises High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Thin Layer Chromatography (TLC), gas-mass spectrometry (GC-MS), liquid-mass spectrometry (LC-MS) and the like, but the methods not only need expensive instruments and equipment, but also have higher requirements on materials to be detected, and can be carried out only by further purification treatment, which can not meet 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 an immunoassay method of the dextropropoxyphene needs to be carried out, and an effective preparation method of the dextropropoxyphene artificial hapten needs to be provided.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a dextropropoxyphene artificial hapten as well as a preparation method and application thereof.

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

in one aspect, the present invention provides a dextropropoxyphene artificial hapten, which has a structure represented by formula I below:

wherein R is selected from substituted or unsubstituted straight chain or branched chain alkylene or aryl, substituted or unsubstituted alkylene or alkyne and substituted or unsubstituted aryl; x is a group from a carboxylic acid, a carboxylic ester, an amine, a maleimide, a halogenated carboxylic acid, a halogenated carboxylic ester, an aldehyde, an alcohol, a dithiopyridine, a vinyl sulfone, a thiocarboxylic acid, or a thiocarboxylic ester.

In the present invention, when the above-mentioned group contains a substituent, the substituent is halogen or C_1-6Alkyl group of (1).

Preferably, R is C_1-6The alkylene group of (3), i.e., R, may be an alkylene group having 1, 2, 3, 4, 5 or 6 carbon atoms.

Preferably, X is a carboxyl group (-COOH), a mercapto group (-SH), or an amino group (-NH)₂)。

Preferably, the dextropropoxyphene artificial hapten is selected from any one of the following dextropropoxyphene hapten derivatives: 2- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) acetic acid (hapten 1); 4- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) butanoic acid (hapten 2); 5- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) pentanoic acid (hapten 3).

In the present invention, the preparation method of the artificial hapten of dextropropoxyphene can be prepared by using dextropropoxyphene as a raw material and adopting a reaction type common in the art, for example, dextropropoxyphene can be firstly removed from a methyl group on a 4-amino to obtain a compound a, and the reaction formula is as follows:

then compound A reacts with a substance containing an-R-X group to obtain the dextropropoxyphene artificial hapten, for example, reacts with carboxylic acid, carboxylic ester, amine, maleimide, halogenated carboxylic acid, halogenated carboxylic ester, aldehyde, alcohol, dithiopyridine, vinyl sulfone, thiocarboxylic acid or thiocarboxylic ester containing an R group to obtain the dextropropoxyphene artificial hapten (when the X group is carboxyl, if the end of the reactant is ester bond, the reactant can be hydrolyzed into carboxylic acid).

In another aspect, the present invention provides a dextropropoxyphene artificial hapten conjugate comprising a dextropropoxyphene artificial hapten and an antigenicity-generating carrier substance conjugated to the dextropropoxyphene artificial hapten.

Preferably, the antigenicity-imparting carrier material comprises a protein, a protein fragment, a protein-fluorescent microsphere conjugate, a protein-nanobead conjugate, a synthetic polypeptide, a semisynthetic polypeptide, cellulose, or an enzyme.

The dextropropoxyphene artificial hapten has no immunogenicity and only has reactogenicity, so the dextropropoxyphene artificial hapten needs to be coupled with a carrier substance, and the host animal is administered with the conjugate to trigger immunogenic response to generate antibodies. Suitable carrier materials typically contain polymeric moieties and include polypeptides, proteins and glycoproteins. Illustrative of useful carrier materials are Bovine Serum Albumin (BSA), Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH), and the like. Alternatively, synthetic poly (amino acids) having a sufficient number of amino groups, such as lysine, may be used, as well as other synthetic or natural polymeric materials having reactive functional groups. Specifically, carbohydrates, yeast or polysaccharides may be combined with haptens to prepare conjugates.

When the artificial hapten of the invention is used for preparing conjugates, if the artificial hapten contains a sulfhydryl group, firstly, an active group such as maleimide, halogen or vinyl sulfone and the like can be introduced into a carrier substance or a marking substance by utilizing a homotype or heterotype bifunctional cross-linking agent, and then the modified carrier substance can be coupled with the sulfhydryl group of the hapten of the invention. Such homo-or hetero-bifunctional crosslinkers are, for example, phenylenedimaleimide, maleimide-succinimidyl ester, N-succinimidyl- (4-iodoacetyl) aminobenzoate, bromoacetyl glycine-N-hydroxysuccinimide, or vinyl sulfone, and the like. For haptens without a thiol group, such as hapten 1, hapten 2 and hapten 3, binding to a carrier substance which has not been previously modified can be carried out by standard binding methods, such as the carbodiimide method or the glutaraldehyde method.

In another aspect, the invention provides the use of a dextropropoxyphene artificial hapten conjugate as described above for the qualitative or quantitative detection of dextropropoxyphene.

In the invention, the dextropropoxyphene artificial hapten conjugate can be used for detecting dextropropoxyphene in a sample, and the sample is contacted with the conjugate during detection; detecting or determining a dextropropoxyphene-bound conjugate; inferring the presence of or specifically determining the amount of dexpropoxyphene in the sample from the calibration curve.

In another aspect, the present invention provides a dexpropoxyphene immunochromatographic assay strip comprising the dexpropoxyphene artificial hapten conjugate as described above.

Preferably, the dextropropoxyphene conjugate is immobilized at a detection zone on a nitrocellulose membrane;

preferably, the dexpropoxyphene immunochromatographic assay test paper sequentially comprises a filter sample paper, a glass cellulose paper, a nitrocellulose membrane and a water absorption paper along a chromatography direction, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line contains the dexpropoxyphene artificial hapten conjugate.

Preferably, the quality control line contains goat anti-mouse IgG polyclonal antibody.

The detection of dexpropoxyphene may employ competitive inhibition. The method can fix the dextropropoxyphene conjugate (such as PPX-BSA) in a detection area on the nitrocellulose membrane, and the dextropropoxyphene small molecule in the sample solution to be detected and the dextropropoxyphene conjugate compete to bind with the anti-dextropropoxyphene antibody marked by functional microspheres (such as colloidal gold, colored latex, fluorescent latex, magnetic beads and the like). The small molecule of the dextropropoxyphene in the sample to be detected can inhibit the combination of the anti-dextropropoxyphene monoclonal antibody and the dextropropoxyphene conjugate, thereby inhibiting the formation of a color band (or a fluorescent band or a magnetic bead band) in a detection area on the nitrocellulose membrane. If a color band (or a fluorescent band or a magnetic bead band) is formed in the detection area after detection, the result is negative, which indicates that the sample to be detected does not contain dextropropoxyphene; on the contrary, if no color band (or fluorescent band or magnetic bead band) is formed, the result is positive, and the detection sample contains dexpropoxyphene.

Usually, an internal quality control is set in the detection. And (3) arranging a quality control area on the nitrocellulose membrane close to the detection area, and fixing the goat anti-mouse IgG polyclonal antibody in the quality control area. In the detection process, no matter whether a sample to be detected contains the dexpropoxyphene or not, functional microsphere markers or other colored marked dexpropoxyphene monoclonal antibodies pre-coated on the glass fiber of the chromatographic carrier can be always combined with goat anti-mouse IgG polyclonal antibodies in a quality control area to form a colored (or fluorescent band or magnetic bead band) quality control band, and the band is a standard for judging whether the chromatographic process is normal or not and whether the detection test paper is deteriorated or not.

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

the synthetic method of the dextropropoxyphene artificial hapten is simple and easy to operate, and the dextropropoxyphene artificial hapten conjugate formed by using the method can be used for qualitative or quantitative detection of dextropropoxyphene, can also be applied to an immunochromatography technology, and has the advantages of high sensitivity, high specificity, simplicity, rapidness, easy operation, no need of any large-scale instrument and equipment and the like.

Drawings

FIG. 1 is a mass spectrum of hapten 1 prepared in example 1.

FIG. 2 is a mass spectrum of hapten 2 prepared in example 2.

FIG. 3 is a mass spectrum of hapten 3 prepared in example 3.

Fig. 4 is a graph showing the results of uv scanning of conjugate a.

Fig. 5 is a graph of the results of the uv scan of conjugate b.

FIG. 6 is a graph showing the results of UV scanning of conjugate c.

Fig. 7 is a schematic structural diagram of the colloidal gold immunochromatographic assay test paper containing the dextropropoxyphene artificial hapten conjugate in the invention, wherein 1: filtering sample paper; 2: a cellophane paper; 3: a detection line (T-line); 4: a quality control line (line C); 5: absorbent paper; 6: a support plate; 7: a nitrocellulose membrane.

FIG. 8 is a schematic diagram of the positions of the sample application hole, the quality control line and the detection line of the colloidal gold immunochromatographic assay test paper containing the dextropropoxyphene artificial hapten conjugate of the present invention and the possibilities of several results of detection.

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.

Haptens 1-3 were synthesized in the following examples, the specific synthetic route of which is as follows:

taking dextropropoxyphene as a starting material, removing methyl on 4-amino, reacting with 2-ethyl bromoacetate under the protection of nitrogen and the catalysis of 18-crown-6-ether, and hydrolyzing a product to obtain 2- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) acetic acid (hapten 1);

using dextropropoxyphene as a starting material, removing methyl on 4-amino, reacting with 4-ethyl bromobutyrate under the protection of nitrogen and the catalysis of 18-crown-6-ether, and hydrolyzing the product to obtain 4- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) butyric acid (hapten 2);

taking dextropropoxyphene as a starting material, removing methyl on 4-amino, reacting with 5-bromovaleric acid ethyl ester under the protection of nitrogen and the catalysis of 18-crown-6-ether, and hydrolyzing a product to obtain 5- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) pentanoic acid (hapten 3).

The detailed synthetic procedure for each hapten is specifically illustrated in examples 1-3 below.

Example 1

Synthesis of 2- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) acetic acid (hapten 1)

Dexpropoxyphene 339.47mg (1mmol) was dissolved in acetonitrile: adding 10mL of water (2:1, V/V), adjusting pH to 5 with formic acid, and dropwise adding new 2M KMnO within 30-35min while stirring₄Aqueous solution 7mL, seal RT reaction for 14 h. Filtering, and adjusting the pH of the filtrate to 7. Extracting with 15mL ethyl acetate for 3 times, combining organic phases, and drying for 4h by anhydrous sodium sulfate; filtering, decompressing and evaporating filtrate to dryness, adding 200.40mg (1.2mmol) of ethyl bromoacetate into the filtrate, placing the mixture into a 25mL three-neck round-bottom flask, adding 10mL of acetonitrile, stirring and dissolving, introducing nitrogen for protection, sequentially adding KI (0.8mmol), catalyst 18-crown-6-ether (0.1mmol) and potassium carbonate (1.2mmol), heating and refluxing for 24h, and tracking and detecting by TLC. After the reaction is finished, the solvent is removed under reduced pressure, 10mL of ice water is added, stirring and dissolving are carried out, extraction is carried out for 3 times by 15mL of ethyl acetate, organic phases are combined, brine is washed twice, the organic phases are collected, and anhydrous sodium sulfate is dried for 4 hours. The mother liquor was collected by filtration, and the solvent was recovered to give 340.98mg of a yellow oil, which was used in the next reaction without purification.

340.98mg of the yellow oily substance was dissolved in 10mL of anhydrous ethanol, 5mL of 20% lithium hydroxide solution was added dropwise thereto, and the mixture was reacted at room temperature for 4 hours. Diluting with 20mL of water, adjusting pH to 6.0, extracting with 20mL of chloroform for 3 times, combining organic phases, washing with water twice, collecting the organic phases, and drying with anhydrous sodium sulfate for 4 h. The solid was removed by filtration, and the solvent was recovered to give 264.26mg of 2- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) acetic acid (hapten 1).

ESI-MS analysis (384.4[ M +1]) (Agilent gas chromatograph 7890A-5975C) was performed on the hapten 1 thus prepared, and the results are shown in FIG. 1, demonstrating that the hapten 1 was synthesized as described above.

Example 2

Synthesis of 4- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) butanoic acid (hapten 2)

Dexpropoxyphene 339.47mg (1mmol) was dissolved in acetonitrile: adding 10mL of water (2:1, V/V), adjusting pH to 5 with formic acid, and dropwise adding new 2M KMnO within 30-35min while stirring₄Aqueous solution 7mL, seal RT reaction for 14 h. Filtering, and adjusting the pH of the filtrate to 7. Extracting with 15mL ethyl acetate for 3 times, and combining the organic phasesDrying for 4 hours by using anhydrous sodium sulfate; filtering, decompressing and evaporating filtrate to dryness, adding 234.06mg (1.2mmol) of ethyl bromobutyrate, placing the mixture into a 25mL three-neck round-bottom flask, adding 10mL of acetonitrile, stirring and dissolving, introducing nitrogen for protection, sequentially adding KI (0.8mmol), catalyst 18-crown-6 (0.1mmol) and potassium carbonate (1.2mmol), heating and refluxing for 24h, and tracking and detecting by TLC. After the reaction is finished, the solvent is removed under reduced pressure, 10mL of ice water is added, stirring and dissolving are carried out, extraction is carried out for 3 times by 15mL of ethyl acetate, organic phases are combined, brine is washed twice, the organic phases are collected, and anhydrous sodium sulfate is dried for 4 hours. The mother liquor was collected by filtration, and the solvent was recovered to give 319.2mg of a yellow oil, which was used in the next reaction without purification.

319.2mg of the yellow oily substance was dissolved in 10mL of anhydrous ethanol, 5mL of 20% lithium hydroxide solution was added dropwise thereto, and the mixture was reacted at room temperature for 4 hours. Diluting with 20mL of water, adjusting pH to 6.0, extracting with 20mL of chloroform for 3 times, combining organic phases, washing with water twice, collecting the organic phases, and drying with anhydrous sodium sulfate for 4 h. The solid was removed by filtration, and the solvent was recovered to give 273.84mg of 4- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) butanoic acid (hapten 2).

ESI-MS analysis (412.5[ M +1]) (Agilent gas chromatograph 7890A-5975C) was performed on the hapten 2 thus prepared, and the results are shown in FIG. 2, which confirmed that the hapten 2 was synthesized as described above.

Example 3

Synthesis of 5- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) pentanoic acid (hapten 3)

Dexpropoxyphene 339.47mg (1mmol) was dissolved in acetonitrile: adding 10mL of water (2:1, V/V), adjusting pH to 5 with formic acid, and dropwise adding new 2M KMnO within 30-35min while stirring₄Aqueous solution 7mL, seal RT reaction for 14 h. Filtering, and adjusting the pH of the filtrate to 7. Extracting with 15mL ethyl acetate for 3 times, combining organic phases, and drying for 4h by anhydrous sodium sulfate; filtering, decompressing and evaporating filtrate to dryness, adding 250.89mg (1.2mmol) of ethyl bromovalerate, placing the mixture into a 25mL three-neck round-bottom flask, adding 10mL of acetonitrile, stirring and dissolving, introducing nitrogen for protection, sequentially adding KI (0.8mmol), catalyst 18-crown-6 (0.1mmol) and sodium methoxide (1mmol), heating and refluxing for 24h, and tracking and detecting by TLC. Inverse directionAfter completion of the reaction, the solvent was removed under reduced pressure, 10mL of ice water was added and dissolved with stirring, the mixture was extracted 3 times with 15mL of ethyl acetate, the organic phases were combined, washed twice with brine, the organic phases were collected, and dried over anhydrous sodium sulfate for 4 hours. The mother liquor was collected by filtration, and the solvent was recovered to give 562.9mg of a yellow oil, which was used in the next reaction without purification.

562.9mg of the yellow oily substance was dissolved in 10mL of anhydrous ethanol, 5mL of 20% lithium hydroxide solution was added dropwise thereto, and the mixture was reacted at room temperature for 4 hours. Diluting with 20mL of water, adjusting pH to 6.0, extracting with 20mL of chloroform for 3 times, combining organic phases, washing with water twice, collecting the organic phases, and drying with anhydrous sodium sulfate for 4 h. The solid was removed by filtration, and the solvent was recovered to give 356.96mg of 5- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) pentanoic acid (hapten 3).

ESI-MS analysis (426.5[ M +1]) (Agilent gas chromatograph 7890A-5975C) was performed on the hapten 3 thus prepared, and the results are shown in FIG. 3, which confirmed that the hapten 3 was synthesized as described above.

Example 4

Hapten 1 is coupled with BSA to obtain a conjugate a

The preparation method comprises the following steps: 50mg of hapten 1 was dissolved in 1mL of DMF, 100mg of EDC hydrochloride and 50mg of NHS were added with stirring and reacted at room temperature for 2 hours, and a solution of 200mg of BSA and 5mL of PBS (0.01mol/L, pH 7.2) prepared in advance was slowly added and reacted at room temperature for 24 hours with stirring. Then dialyzed against PBS (0.01mol/L, pH 7.4) at 4 ℃ for 3 days (6 changes), centrifuged, the supernatant was collected and lyophilized to give conjugate a.

The obtained conjugate a was subjected to ultraviolet scanning (Thermo micro ultraviolet-visible spectrophotometer NanoDrop 2000C), and as a result, as shown in FIG. 4, it can be seen that BSA has typical absorptions at 279nm and 252nm, and absorptions of hapten 1 and conjugate a have distinct and shifted at 279nm and 252nm, which indicates that the artificial antigen conjugate has different ultraviolet absorption characteristics from its precursor substance, indicating that hapten and carrier protein are successfully coupled, and according to the report of old establishment of literature, Chenmei, Zhaojie et al.

Example 5

Hapten 2 was conjugated to BSA to give conjugate b, in the same manner as in example 4 except that hapten 1 was replaced by hapten 2.

The ultraviolet scanning spectrum of the conjugate b is shown in FIG. 5, and it can be seen that BSA has typical absorption at 279nm and 252nm, and the absorption of hapten 2 and conjugate b has obvious and shifted at 279nm and 252nm, which indicates that the artificial antigen conjugate has different ultraviolet absorption characteristics with its precursor substance, and indicates that hapten 2 is successfully conjugated with carrier protein.

Example 6

Hapten 3 was coupled to BSA to give conjugate c, in the same manner as in example 4 except that hapten 1 was replaced by hapten 3.

The ultraviolet scanning spectrum of the conjugate c is shown in FIG. 6, and it can be seen that BSA has typical absorption at 279nm and 252nm, and the absorption of hapten 3 and conjugate c has obvious and shifted at 279nm and 252nm, which indicates that the artificial antigen conjugate has different ultraviolet absorption characteristics with its precursor substance, and indicates that the conjugation of hapten 3 and carrier protein is successful.

Example 7

Preparation of colloidal gold immunochromatographic assay test paper for detecting dextropropoxyphene

1. Preparation of colloidal gold-monoclonal antibody conjugate glass fiber strip

The colloidal gold-labeled dexpropoxyphene antibody provided by guangzhou mobil biotechnology, Inc. was mixed with 10mM phosphate buffer solution having pH of 7.4 to prepare a solution having a concentration of 0.5mg/mL, and the solution was uniformly coated on a glass cellulose paper in a coating amount of 50. mu.l/cm²And vacuum drying for 12 h.

2. Preparation of detection line and quality control line

Detection line (T line): conjugate a was mixed with 10mM phosphate buffer solution having pH 7.4 to prepare a solution having a concentration of 0.2mg/mL, which was sprayed on a nitrocellulose membrane.

Quality control line (line C): goat anti-mouse IgG polyclonal antibody was mixed with 10mM phosphate buffer pH 7.4 to prepare a solution of 0.1mg/mL, and sprayed onto nitrocellulose membrane.

Then dried at 50 ℃ for 12 h.

3. Assembly of colloidal gold immunochromatographic assay test paper

Sequentially overlapping and assembling the sample filtering paper, the glass cellulose paper coated with the colloidal gold labeled dextropropoxyphene antibody, the nitrocellulose membrane coated with the detection line (T line) and the quality control line (C line) and the absorbent paper to form a test strip, placing the test strip on a support plate, and fixing by using glue, as shown in figure 7, wherein 1: filtering sample paper; 2: a cellophane paper; 3: a detection line (T-line); 4: a quality control line (line C); 5: absorbent paper; 6: a support plate; 7: a nitrocellulose membrane.

Example 8

Test of colloidal gold immunochromatographic assay test paper for detecting dextropropoxyphene

1. Sample preparation

According to the test requirements, a blank urine sample is added with a dextromethorphan standard and respectively prepared into samples containing dextromethorphan, and the concentrations of the samples are 0ng/mL, 50ng/mL, 75ng/mL, 100ng/mL, 125ng/mL, 150ng/mL, 175ng/mL, 200ng/mL, 225ng/mL, 250ng/mL, 275ng/mL, 300ng/mL, 325ng/mL, 350ng/mL, 375ng/mL and 400 ng/mL.

2. Detection and results

2.1, carrying out quantitative gradient test on the series of concentrations, wherein a sample is added to a sample adding hole (shown in figure 8) on a test strip during the test, and both a detection line and a quality control line on the test strip are negative during the test; the detection line does not develop color, and the quality control line develops color to be positive; the detection line is colored, the quality control line is not colored, or the detection line and the quality control line are not colored, and the test fails; the results of the sensitivity test in this example are shown in Table 1.

TABLE 1 test results of sensitivity of the test paper for immunochromatography analysis of colloidal gold

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

The test result shows that the minimum detection quantity of the test paper is 150 ng/mL.

2.2 negative samples

According to the test requirements, 426 negative samples subjected to GC/MS analysis tests are respectively loaded on the colloidal gold immunochromatographic assay test paper for detecting dextropropoxyphene, and the results are negative.

Example 9

Drug crossover experiment of colloidal gold immunochromatographic assay test paper for detecting dextropropoxyphene

88 drugs and common drugs, respectively, are prepared with negative urine to prepare samples with the concentration of 100 mul/mL. The color development was judged according to the colorimetric card of Guangzhou Wanfu Biotechnology Co., Ltd., the negative result was represented by "-" and the positive result was represented by "+".

TABLE 2 Cross-over test results of the colloidal gold immunochromatographic assay test paper

The colloidal gold immunochromatographic assay test paper for detecting dexpropoxyphene of the embodiment is prepared by a conventional preparation method and comprises a test strip and a support plate for supporting the test strip; the test strip is formed by sequentially overlapping sample filtering paper, a chromatographic material, a nitrocellulose membrane and absorbent paper, wherein the chromatographic material is pre-coated with a dextropropoxyphene monoclonal antibody or a polyclonal antibody which is labeled by colloidal gold or colored labels. Adsorption detection lines and quality control lines are arranged on the nitrocellulose membrane; the detection line is a dextropropoxyphene conjugate, and the area where the detection line is located is a detection area; the quality control line is a goat anti-mouse polyclonal antibody, and the area where the quality control line is located is a quality control area.

Example 10

Preparation of fluorescent latex immunochromatographic assay test paper for detecting dextropropoxyphene

1. Preparation of fluorescent latex-monoclonal antibody conjugate glass fiber strip

Fluorescent latex-labeled dexpropoxyphene antibody supplied by guangzhou mobil biotechnology limited was mixed with 10mM phosphate buffer solution having a pH of 7.8 to prepare a solution having a concentration of 0.5mg/mL, and the solution was uniformly coated on a glass cellulose paper in an amount of 40. mu.l/cm²And vacuum drying for 12 h.

2. Preparation of detection line and quality control line

And (3) detection line: the conjugate b was mixed with 10mM phosphate buffer solution having pH 7.8 to prepare a solution having a concentration of 0.5mg/mL, which was sprayed on a nitrocellulose membrane.

Quality control line: goat anti-mouse IgG polyclonal antibody was mixed with 10mM phosphate buffer solution having pH 7.8 to prepare a solution having a concentration of 0.4mg/mL, and sprayed on a nitrocellulose membrane.

Then dried at 50 ℃ for 12 h.

3. Assembly of fluorescent latex immunochromatographic assay test paper

The assembly method was identical to example 7.

Example 11

Sensitivity test of fluorescence latex immunochromatographic assay test paper for detecting dextropropoxyphene

1. Sample preparation:

the process was in accordance with example 8

2. Detection and results

The above series of concentrations were tested in quantitative gradient and the results of the sensitivity experiments are shown in table 3.

TABLE 3 sensitivity test results of the fluorescence latex immunochromatographic assay test paper

Concentration (ng/mL)	0	50	75	100	125	150	175	200
									Test paper results	-	-	+	+	+	+	+	+
Concentration (ng/mL)	225	250	275	300	325	350	375	400
									Test paper results	+	+	+	+	+	+	+	+

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

The test result shows that the minimum detection amount of the test paper is 75 ng/mL.

Example 12

Preparation of color latex immunochromatographic assay test paper for detecting dextropropoxyphene

1. Preparation of color latex-monoclonal antibody conjugate glass fiber strip

Color latex-labeled dexpropoxyphene antibody supplied by guangzhou mobil biotechnology, Inc. was mixed with 10mM phosphate buffer solution having pH of 7.8 to prepare a solution having a concentration of 0.3mg/mL, and the solution was uniformly coated on a glass cellulose paper in an amount of 40. mu.l/cm²And vacuum drying for 12 h.

2. Preparation of detection line and quality control line

And (3) detection line: conjugate c was mixed with 10mM phosphate buffer solution having pH 7.8 to prepare a solution having a concentration of 0.5mg/mL, and sprayed on a nitrocellulose membrane.

Then dried at 50 ℃ for 12 h.

3. Assembly of color latex immunochromatographic assay test paper

The assembly method was identical to example 7.

Example 13

Sensitivity test of color latex immunochromatographic assay test paper for detecting dextropropoxyphene

1. Sample preparation:

the process was in accordance with example 8

2. Detection and results

The above series of concentrations were tested in quantitative gradient and the results of the sensitivity experiments are shown in table 4.

TABLE 4 sensitivity test results of the color latex immunochromatographic assay test paper

Concentration (ng/mL)	0	50	75	100	125	150	175	200
									Test paper results	-	-	-	-	-	+	+	+
Concentration (ng/mL)	225	250	275	300	325	350	375	400
									Test paper results	+	+	+	+	+	+	+	+

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

The applicant states that the present invention is illustrated by the above examples to the dexpropoxyphene artificial antigen of the present invention and the preparation method and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must rely on the above examples to be implemented. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A dexpropoxyphene artificial hapten, wherein the dexpropoxyphene artificial hapten has the structure shown in the following formula I:

2. The dextropropoxyphene artificial hapten according to claim 1, wherein R is C_1-6An alkylene group of (a).

3. The dextropropoxyphene artificial hapten according to claim 1 or 2, wherein X is a carboxyl group, a thiol group or an amino group.

4. A dextropropoxyphene artificial hapten according to any one of claims 1-3, wherein the dextropropoxyphene artificial hapten is selected from any one of the following dextropropoxyphene hapten derivatives:

2- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) acetic acid;

4- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) butanoic acid;

5- (((2R,3S) -3-benzyl-4-ethoxy-2-methyl-4-oxo-3-phenylbutyl) (methyl) amino) pentanoic acid.

5. A dexpropoxyphene artificial hapten conjugate comprising a dexpropoxyphene artificial hapten as claimed in any one of claims 1-4 and an antigenic generating carrier substance conjugated to the dexpropoxyphene artificial hapten.

6. The dextropropoxyphene artificial hapten conjugate according to claim 5, wherein the antigenicity-conferring carrier material comprises a protein, a protein fragment, a synthetic polypeptide, a semi-synthetic polypeptide, cellulose or an enzyme.

7. Use of a dextropropoxyphene artificial hapten conjugate according to claim 5 or 6 for the qualitative or quantitative detection of dextropropoxyphene.

8. A dexpropoxyphene immunochromatographic assay strip comprising the dexpropoxyphene artificial hapten conjugate of claim 5 or 6.

9. The dexpropoxyphene immunochromatographic assay test strip of claim 8, wherein the dexpropoxyphene conjugate is immobilized on a detection zone on a nitrocellulose membrane.

10. The dexpropoxyphene immunochromatographic assay test paper according to claim 8 or 9, which comprises a filter sample paper, a glass cellulose paper, a nitrocellulose membrane and a water absorbent paper in sequence along a chromatography direction, wherein the nitrocellulose membrane is provided with a detection line and a quality control line, and the detection line contains the dexpropoxyphene artificial hapten conjugate;