CN112724029A - Methamphetamine hapten, artificial antigen, antibody and application thereof - Google Patents
Methamphetamine hapten, artificial antigen, antibody and application thereof Download PDFInfo
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- CN112724029A CN112724029A CN202011486023.XA CN202011486023A CN112724029A CN 112724029 A CN112724029 A CN 112724029A CN 202011486023 A CN202011486023 A CN 202011486023A CN 112724029 A CN112724029 A CN 112724029A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
- C07C229/02—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton
- C07C229/04—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated
- C07C229/06—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton
- C07C229/10—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings
- C07C229/12—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino and carboxyl groups bound to acyclic carbon atoms of the same carbon skeleton the carbon skeleton being acyclic and saturated having only one amino and one carboxyl group bound to the carbon skeleton the nitrogen atom of the amino group being further bound to acyclic carbon atoms or to carbon atoms of rings other than six-membered aromatic rings to carbon atoms of acyclic carbon skeletons
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/946—CNS-stimulants, e.g. cocaine, amphetamines
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
Abstract
The invention relates to the technical field of methamphetamine detection, and particularly relates to a methamphetamine hapten, an artificial antigen, an antibody and application thereof. Hapten of methamphetamine, which is a compound shown in formula I. The invention aims at methamphetamine, prepares methamphetamine carboxyl hapten MET-3 and couples different carrier proteins as immunogen, the prepared antibody aiming at the methamphetamine has less than 5 percent of cross with the amphetamine, and the antibody has structural analogue of ephedrine,Methoxyphenamine, common drugs morphine and ketamine have no obvious cross reaction, the established enzyme-linked immunoassay method can realize the specific detection of methamphetamine, has higher accuracy, is convenient and quick, and provides a quick and efficient detection means for detecting the methamphetamine in samples such as hair and the like.
Description
Technical Field
The invention relates to the technical field of methamphetamine detection, and particularly relates to a methamphetamine hapten, an artificial antigen, an antibody and application thereof.
Background
Methamphetamine (C)10H15N) is a widely abused neurostimulant. The methamphetamine can enter a human body by oral administration, hot inhalation, direct nasal inhalation, intravenous injection and the like, and has high bioavailability. Methamphetamine is an indirect agonist of dopamine and norepinephrine in the central nervous system, and long-term intake of methamphetamine can cause abnormal brain function and cause psychopathic symptoms such as auditory hallucination, abnormal excitement, pernicious paradoxus, cognitive disorder and the like. On the other hand, methamphetamine produces a series of physiological effects mainly through indirect stimulation of neurotransmitters, which cause damage to neurons, and also make the abuse of methamphetamine easy to addict and difficult to quit. In view of the human body damage and the social hazard of the methamphetamine, the methamphetamine is listed as a controlled drug in China and is not abused.
After the methamphetamine is sucked, the concentration peak value is reached in a human body within about several minutes to 3 hours, the half-life period in plasma is about 10 hours, and the half-life period in urine is about 25 hours, so that in a body fluid test material, the drug metabolism is fast, and the detection can not be carried out after 2-4 days generally. Compared with body fluid test materials, after being drunk by ice, methamphetamine can accumulate in hair for a long time, and the 3cm hairy root close to the scalp can reflect the drug-taking history of 6 months, so the hair test materials become the most commonly used biological test materials in judicial identification. The hair sample has the advantages of easy sampling, stable detection, long detection period and the like, and is the most common biological sample in judicial identification. The establishment of the rapid high-throughput screening method for the methamphetamine in the hair has important significance for fighting against crimes and suppressing drug abuse, and simultaneously meets the requirements of safe physical examination of special high-risk occupational drug involvement. The enzyme-linked immunoassay technology is an analysis method based on antigen-antibody specific reaction, has the characteristics of sensitivity, rapidness, high throughput and the like, and can be matched with an instrument confirmation technology to meet the requirement of rapid drug screening. An enzyme-linked immunosorbent assay (ELISA) method for researching methamphetamine in hair has very important economic and social significance for fast screening and detecting large-scale samples of drugs.
Disclosure of Invention
The present invention relates to compounds of formula I:
according to a second aspect of the invention, it relates to methamphetamine artificial antigens which are conjugates of a compound as described above with an immunogenic carrier.
Optionally, the methamphetamine artificial antigen as described above, having the formula shown in formula II:
optionally, the methamphetamine artificial antigen as described above, wherein the immunogenic carrier is at least one selected from the group consisting of keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulin, hormone, and ovalbumin.
According to a third aspect of the invention, an antibody is provided, which is obtained by immunizing methamphetamine artificial antigen as described above.
According to a fourth aspect, the invention relates to a methamphetamine detection kit or test strip containing the antibody.
Optionally, the kit as described above, further comprising one or more of methamphetamine conjugated to a signal substance or the compound of claim 1, a solid support, a reaction buffer, a blocking solution, a wash solution, a positive control stimulus, and a negative control.
Optionally, the kit as described above, wherein the antibody is immobilized and coated on the solid phase carrier.
Optionally, the kit as described above, wherein the solid support is selected from the group consisting of a test tube, an EP tube, a multiwell plate, a microplate well, and a microsphere.
Optionally, the kit as described above, wherein the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, and an enzyme.
According to a fifth aspect of the present invention, the present invention also relates to the use of the antibody as described above or the kit or test strip as described above for detecting methamphetamine.
Alternatively, for use as described above, the sample to be tested is blood, urine or hair.
The invention has the beneficial effects that:
the invention aims at the methamphetamine, prepares the methyl amphetamine carboxyl type hapten MET-3 and couples different carrier proteins as immunogens, the prepared antibody aiming at the methamphetamine has less than 5 percent of cross with the amphetamine, has no obvious cross reaction on structural analogues of ephedrine, methoxamine, common narcotic morphine and ketamine, and the established enzyme-linked immunoassay method can realize the specificity detection of the methamphetamine, has higher accuracy, is convenient and quick, and provides a quick and efficient detection means for detecting the methamphetamine in samples such as hair and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a chart of mass spectrometric identification of MET-3 in one embodiment of the present invention;
FIG. 2 shows the result of detecting a characteristic absorption peak of a carrier protein according to an embodiment of the present invention;
fig. 3 is a standard curve of methamphetamine in one embodiment of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The present invention relates to compounds of formula I:
the compound can be used as a hapten of methamphetamine, the hapten furthest reserves the structural characteristics of a functional group of the methamphetamine, and the methamphetamine artificial antigen prepared by the hapten can exert the optimal structure-effect advantage, so that the antibody with strong specificity and higher sensitivity is obtained.
The term "hapten" refers to a partial or incomplete antigen. Haptens are protein-free substances that do not stimulate antibody formation but react with antibodies.
The invention also relates to a methamphetamine artificial antigen which is a conjugate of the compound and an immunogenic carrier.
In some embodiments, the methamphetamine artificial antigen has the formula shown in formula II:
as used herein, an "immunogenic carrier" is an immunogenic substance, typically a protein, that is capable of conjugating haptens at one or more positions, thereby enabling the production of antibodies that bind to these haptens. Examples of immunogenic carrier materials include, but are not limited to, proteins, glycoproteins, complex polyaminopolysaccharides, particles, and nucleic acids that are recognized as foreign and thereby elicit an immune response in a host. The polyaminopolysaccharides may be prepared from the polysaccharides using any conventional method known for such preparation.
Various protein types may be used as immunogenic carriers, and in some preferred embodiments, the carrier protein is selected from at least one of keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulins, hormones (e.g., insulin), and ovalbumin.
The immunogenic carrier may also include polyaminopolysaccharides, which are high molecular weight polymers constructed by repeated condensation of monosaccharides. Examples of polysaccharides are starch, glycogen, cellulose, carbohydrates, gums such as gum arabic, agar, and the like. The polysaccharide further comprises poly (amino acid) residues and/or lipid residues.
The immunogenic carrier can also be a poly (nucleic acid) alone or conjugated to one of the above poly (amino acids) or polysaccharides.
The immunogenic carrier may also comprise solid particles. The particles typically have a diameter of at least about 0.02 micrometers (μm) and no more than about 100 μm, and typically from about 0.05 μm to 10 μm. The particles may be organic or inorganic, swellable or non-swellable, porous or non-porous, optimally close to the density of water, typically about 0.7 to 1.5g/mL, and are composed of materials that may be transparent, partially transparent or opaque. The particles may be biological material such as cells and microorganisms, including non-limiting examples such as erythrocytes, leukocytes, lymphocytes, hybridomas, streptococci (Streptococcus), Staphylococcus aureus (Staphylococcus aureus), escherichia coli (e. The particles may also comprise organic and inorganic polymers, liposomes, latex, phospholipid vesicles or lipoproteins.
The invention also provides an antibody which is obtained by immunizing the methamphetamine artificial antigen.
The term "antibody" refers to a specific protein capable of binding to an antigen or a part thereof (capable of binding to an antipsychotic drug or a metabolite thereof according to the invention). Antibodies are produced in response to an immunogen introduced into a host, such as an animal or human, by injection. The antibody may be a monoclonal antibody or a polyclonal antibody, or may be an antibody fragment thereof.
"antibody" refers to an intact antibody or a fragment thereof that competes for binding with an intact antibody. Generally, an antibody can be said to specifically bind an antigen when the dissociation constant is less than or equal to 1 μ M, preferably less than or equal to 100nM, and most preferably less than or equal to 10 nM. An antibody fragment comprises a portion of an intact antibody, preferably the antigen binding or variable region of an intact antibody. Binding fragments include Fab, Fab ', F (ab')2 and Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments. An antibody other than a "bispecific" or "bifunctional" antibody is understood to be identical at each of its binding sites.
Antibodies can be prepared by any of a variety of techniques known to those of ordinary skill in the art. In one such technique, an immunogen comprising a methamphetamine artificial antigen is initially injected into any of a variety of mammals (e.g., mice, rats, rabbits, sheep, and goats). The methamphetamine artificial antigen can also be combined with an immunologic adjuvant (such as Freund's complete adjuvant and Freund's incomplete adjuvant) to inject the immunogen into an animal host; more preferably, the injections are administered according to a predetermined schedule of multiple booster immunizations and the animals are bled periodically. Antibodies specific for the polypeptide can then be purified from serum or like components, for example, by affinity chromatography using the polypeptide attached to a suitable solid support.
Monoclonal antibodies can be generated by the fully established hybridoma method of Kohler and Milstein, e.g., Nature256:495-497 (1975). Hybridoma methods generally involve immunizing a host or lymphocytes from the host, harvesting lymphocytes that secrete lymphocytes or monoclonal antibodies with the potential to secrete lymphocytes, fusing the lymphocytes with immortalized cells, and selecting cells that secrete the desired monoclonal antibodies.
Lymphocytes can be fused with immortalized cell lines to form hybridoma cells, and the use of a fusing agent such as polyethylene glycol can facilitate this process. By way of illustration, mutant rodent, bovine or human myeloma cells immortalized by transformation may be used. In contrast to unfused immortalized cells, a population of substantially pure hybridoma cells is preferred. Thus, after fusion, the cells can be grown in a suitable medium that inhibits the growth or survival of unfused, immortalized cells, for example, by using mutant myeloma cells that lack hypoxanthine guanine phosphoribosyl transferase (HGPRT). In such examples, hypoxanthine, aminopterin, and thymidine can be added to the medium (HAT medium) to prevent the growth of HGPRT-deficient cells while allowing the growth of hybridomas.
The invention also relates to a methamphetamine detection kit containing the antibody.
In some embodiments, the kit further comprises one or more of methamphetamine coupled to a signal substance or the compound of claim 1, a solid support, a reaction buffer, a blocking solution, a wash solution (e.g., PBS, etc.), a positive control stimulus, and a negative control, and further comprises one or more of a compound, a solid support, a reaction buffer, a blocking solution, a wash solution (e.g., PBS, etc.), a methamphetamine standard, and a negative control, as described above.
In some embodiments, the kit also contains a chromogenic reagent for the signal substance (e.g., if the signal substance is horseradish peroxidase, then the chromogenic reagent is a TMB developing solution or ECL).
In some embodiments, the antibody is immobilized coated on the solid support.
In some embodiments, the solid support is selected from a test tube, an EP tube, a multi-well plate, a microplate well, or a microsphere.
In the present invention, the term "microparticle" may be a sphere, a nearly sphere, a cube, a polyhedron, or an irregular shape. The diameter of the microspheres is preferably 10nm to 1mm, for example 100nm, 500nm, 1 μm, 10 μm, 100 μm, 500 μm; preferably 400nm to 10 μm.
The fine particles are preferably magnetic fine particles, and the composition thereof contains a magnetic substance. The magnetic substance may be a metal (simple metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. Metals such as iron, alnico, and the like; non-metals, e.g. ferrite non-metals (preferably Fe)2O3Or Fe3O4Magnetic nanoparticles); a composite of metal and non-metal such as neodymium iron boron rubber magnetic composite.
The multiwell plate is preferably an elisa plate, and may contain 8, 16, 32, 48, 64, 96 or more wells.
In some embodiments, the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, or an enzyme.
The following non-limiting section lists these markers:
enzymes which produce a detectable signal, e.g.by colorimetry, fluorescence or luminescence, such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase and glucose-6-phosphate dehydrogenase.
Chromophores such as fluorescence, quantum dots, fluorescent microspheres, luminescent compounds (e.g. acridinium esters) and dyes.
Groups with electron density that can be detected by electron microscopy or by its electrical properties, such as conductivity, amperometry, voltage measurement and resistance.
A detectable group, such as one whose molecular size is sufficient to induce a detectable modification in its physical and/or chemical properties; such detection can be achieved by optical methods (e.g., diffraction, surface plasmon resonance, surface variation and angle of contact variation) or physical methods (e.g., atomic spectroscopy and tunneling).
Electron-dense substances, e.g. radioactive molecules (e.g. of the type32P，35S or125I)。
The invention also relates to the application of the antibody or the kit in detecting methamphetamine.
In some embodiments, the sample to be tested is blood, serum, plasma, anticoagulation, cell culture supernatant, saliva, semen, amniotic fluid, villi, tissue or tissue lysate, pharyngeal swab, nasal swab, conjunctival swab, stool specimen, stool, urine, bronchial lavage, alveolar lavage, sputum. Preferred samples are blood, urine or hair.
The antibody and the enzyme-linked immunoassay method can be used for the specificity detection of the methamphetamine residue in the hair, and the method is used for the IC of the methamphetamine501.00ng/mL, linear detection range (IC)20-IC80) 0.22-4.46ng/mL, detection limit (IC)10) 0.13ng/mL, and a cross-reactivity with amphetamine of 4.2%. The antibody has the obvious advantages of high sensitivity, good accuracy, simple and quick sample pretreatment method and the like, so that the antigen and the antibody provided by the invention can be used for establishing an enzyme-linked immunoassay method of methamphetamine. The method is stable and reliable, has low cost, and has important significance for the research and development of the methamphetamine rapid detection kit and the colloidal gold test strip.
Embodiments of the present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of haptens
S1, 70mg of methamphetamine and 101.9mg of ethyl bromopropionate are placed in a 25mL three-neck round-bottom flask, 15mL of acetonitrile is added and stirred to be dissolved, nitrogen is introduced for protection, 62.3mg of KI, 12.4mg of catalyst 18-crown-6 and 77.8mg of potassium carbonate are sequentially added, heating reflux reaction is carried out for 24 hours, and tracking detection is carried out 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 using 30mL 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. Filtering to collect mother liquor, recovering solvent to obtain yellow oily MET-1, purifying with silica gel column (methanol/chloroform, 1: 15, V/V), and storing at-20 deg.C.
Reaction formula 1:
s2, dissolving 50mg MET-1 and 23.9mg amphetamine in acetonitrile, adding 23.1mg anhydrous potassium carbonate, refluxing at 70 ℃ for 6h, and detecting by TLC. After completion of the reaction, the solvent was distilled off under reduced pressure, and then extracted with ethyl acetate/saturated brine 3 times, and the organic phases were combined and dried over anhydrous sodium sulfate for 4 hours. The mother liquor was collected by filtration and rotary evaporated under reduced pressure, and the crude MET-2 product was purified by silica gel column (methanol/dichloromethane, 1: 15, V/V).
Reaction formula 2:
s3, dissolving 50mg of MET-2 and 19.7mg of succinic anhydride in 20mL of acetonitrile, introducing nitrogen for protection, sequentially adding 11.5mg of KI, 3.7mg of catalyst 18-crown-6 and 23.4mg of potassium carbonate, heating for reflux reaction for 24 hours, and performing TLC tracking detection. After the reaction is finished, the solvent is removed under reduced pressure, 10mL of ice water is added, stirring and dissolving are carried out, ethyl acetate is used for 3 times, organic phases are combined, brine is used for washing twice, the organic phases are collected, anhydrous sodium sulfate is used for drying for 4 hours, mother liquor is filtered and collected, and then a silica gel column is used for purifying to obtain the target hapten MET-3 (methanol/dichloromethane, 1: 15, V/V), wherein FIG. 1 is a MET-3 mass spectrum identification chart.
Reaction formula 3:
EXAMPLE 2 preparation of Artificial vector
1. Preparation of immunogen MET-3-BSA
15.0mg of MET-3 hapten was dissolved in 0.30mL of N, N-dimethylformamide, and 9.87mg of N, N-dicyclohexylcarbodiimide and 5.50mg of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 4 ℃ for reaction overnight, and after centrifugation, the supernatant was counted as solution A. Dissolving 26.48mg BSA in 6mL PBS (0.01 mol/L), marking as solution B, dropping solution A into solution B under stirring, reacting for 12h at 4 ℃ in a dark place, centrifuging after the reaction is finished, taking supernatant, dialyzing the PBS solution at 4 ℃ for 3 days to obtain methamphetamine artificial immunogen, subpackaging the methamphetamine artificial immunogen in 1mL centrifuge tubes at the concentration of 1mg/mL, and freezing the obtained methamphetamine artificial immunogen in a refrigerator at-20 ℃ for later use.
2. Preparation of enzyme-labeled antigen MET-3-HRP
15.0mg of MET-3 hapten was dissolved in 0.30mL of N, N-dimethylformamide, and 9.87mg of N, N-dicyclohexylcarbodiimide and 5.50mg of N-hydroxysuccinimide were added thereto, and the mixture was stirred at 4 ℃ for reaction overnight, and after centrifugation, the supernatant was counted as solution A. Dissolving 17.54mg of HRP in 6mL of 0.01mol/L PBS (phosphate buffer solution), marking as solution B, dripping the solution A into the solution B while stirring, reacting for 12h at 4 ℃, centrifuging after the reaction is finished, taking supernatant, dialyzing for 3 days by using the PBS solution at 4 ℃ to obtain the methamphetamine enzyme-labeled antigen, subpackaging in 1mL of centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use.
3. Characterization of Methamphetamine immunogens and enzyme-labeled antigens
(1) The immunogen and the enzyme-labeled antigen are identified by adopting an ultraviolet scanning method, measuring the ultraviolet absorption spectrum of the immunogen and the coating antigen within the wavelength range of 180-350 nm, comparing the scanning curves of different substances, and identifying whether the coupling of the hapten and the carrier protein is successful. Because both the carrier protein and the hapten have maximum absorption peaks under the ultraviolet spectrum condition, if the coupling is successful, the characteristic absorption peaks are mutually superposed, thereby causing the blue shift of the maximum absorption peak.
(2) As shown in figure 2, the characteristic absorption peak of the carrier protein at about 280nm shows obvious blue shift, so that the successful coupling of the immunogen and the enzyme-labeled antigen can be proved from the ultraviolet spectrum scanning result.
EXAMPLE 3 Methamphetamine polyclonal antibody preparation
1. Animal immunization
When 2-2.5 kg of New Zealand white rabbits are immunized for the first time, 1.0mL of immunogen is injected, 0.5mL of immunogen with the concentration of 1mg/mL is taken, an equal volume of Freund complete adjuvant is added, and after sufficient emulsification, subcutaneous multi-point injection is carried out on the backs of the white rabbits, wherein each point is about 200 muL; after 21 days, 2 nd immunization is carried out, the immunization dose is 1.0 mL/mouse, and the 2 nd immunization dose is emulsified by Freund incomplete adjuvant; booster immunizations were performed 1 time, typically 5 times, every 14 days thereafter. After the 3 rd and 5 th immunizations, blood was collected for antiserum titer determination. The experimental animals are numbered, managed and recorded, and the health conditions of the white rabbits are noticed.
2. Antiserum effect assay
(1) On the 7 th day after 3 rd and 5 th immunization, 40 μ L of experimental white rabbit ear vein blood is collected, antiserum is obtained by centrifugation, the antibody concentration is detected by nanodrop, and the antiserum is subpackaged at-20 ℃ for standby.
(2) The titer and specificity of antiserum are determined by enzyme-linked immunosorbent assay, which comprises the following steps:
s1, coating: the antiserum at 1mg/mL was diluted 1000-fold with carbonate buffer and used as a blank, 100. mu.L/well was added to the microplate, and the microplate was incubated overnight in a 37 ℃ water bath.
S2, washing: and (3) pouring out liquid in the holes, setting parameters of a plate washing machine, adding 300 mu L of deionized water into each hole, washing the plate for 2 times, and then spin-drying the washing liquid.
S3, sealing: adding 120 μ L of sealing liquid into each hole, sealing at 37 deg.C for 3 hr, spin-drying the liquid in the hole, and oven-drying in an oven at 37 deg.C for 1 hr.
S4, adding an enzyme-labeled antigen and a standard substance: diluting enzyme-labeled antigen in a gradient manner; dissolving the methamphetamine standard substance in a proper amount of methanol to prepare a standard substance solution of 1.0mg/mL, and storing at 4 ℃ for later use. The potency is listed as: adding 100 mu L of blank diluent and 100 mu L of enzyme-labeled antigen diluted in a gradient manner into each hole, and finally adding PBS into the two holes to serve as blank control; inhibition column: adding 20 mu L of standard solution and 100 mu L of enzyme-labeled antigen diluted in a gradient manner into each hole, and finally adding the standard dilution solution into the two holes to serve as blank control; after shaking, incubation was carried out at room temperature for 40min and the plate was washed 6 times.
S5, color development: adding 100 μ L of TMB developing solution into each well, developing at room temperature for 20min, and adding 100 μ L of stop solution (10% H) into each well2SO4)。
S6, reading measurement: the absorbance (OD) was read with a microplate reader at a wavelength of 450 nm.
(3) The antiserum dilution multiple with the absorbance value within the range of 1.0-1.5 is selected as the antiserum titer, the antiserum effect is obtained from the inhibition rate of the antiserum, and under the same drug concentration, the higher the inhibition rate is, the higher the sensitivity of the antibody to the drug is.
3. Purification of antisera
(1) Selecting experimental white rabbits with the best inhibition rate and potency, collecting whole blood from hearts after anesthesia and coma, and carrying out euthanasia treatment on experimental animals through spinal dislocation;
(2) after the blood is subjected to warm bath at 37 ℃ for 20min, centrifuging at 5000rpm for 20min, taking the upper layer serum, subpackaging and storing at-20 ℃;
(3) diluting 5mL of rabbit antiserum with 10mL of acetate buffer solution, and adjusting the pH value to 4.5 by using 1mol/L NaOH solution;
(4) slowly and dropwise adding 375 mu L of n-caprylic acid into the serum under the stirring condition, continuously stirring for 30min, and standing for 1h at the temperature of 4 ℃ to fully precipitate and separate out the foreign protein;
(5) centrifuging at 4 deg.C and 12000rpm for 15min, and filtering with 125 μm filter membrane to obtain supernatant;
(6) adding 1.5mL of 0.1mol/L PBS buffer solution into the supernatant, and adjusting the pH value by using a 1mol/L NaOH solution to ensure that the pH value is 7.4;
(7) 4.432g of ammonium sulphate was added slowly over 30min under ice-bath conditions to enable 45% saturation; standing at 4 deg.C for 2 hr, centrifuging at 4 deg.C at 12000rpm for 15min, removing supernatant, and collecting precipitate;
(8) the precipitate was redissolved in 5mL of 0.1mol/L PBS (pH 7.4) and ultrafiltered 3 times;
(9) the polyclonal antibody after ultrafiltration was diluted 10-fold with 0.1mol/L PBS (pH 7.4), dispensed, and stored at-20 ℃.
EXAMPLE 4 establishment of method for enzyme-linked immunoassay of methamphetamine in Hair
1. The enzyme-linked immunosorbent assay (ELISA) reaction specifically comprises the following steps:
s1, coating: the methamphetamine antibody is diluted to a proper concentration by carbonic acid buffer solution, added into a hole of an enzyme-labeled plate, and put in a water bath cabinet at 37 ℃ overnight at a rate of 100 mu L/hole.
S2, washing: and (4) pouring out liquid in the holes, washing the plate for 2 times, adding 300 mu L of washing liquid into each hole, and drying by spin.
S3, sealing: adding 120 μ L of sealing solution into each well, sealing at 37 deg.C for 3 hr, drying, and placing in oven at 37 deg.C for 1 hr.
S4, pretreatment of hair: 25mg of hair are weighed into a milling tube, 1mL of PBS (pH 8.0) and 20 zirconium beads 2mm in diameter are added, the tube is placed in a milling apparatus and shaken at 2800rpm for 25 seconds for 4 cycles with 10 seconds between each cycle. Standing for 30 seconds after the oscillation is finished, and obtaining a supernatant as a sample solution to be detected.
S5, sample adding and incubation: sequentially adding 20 mu L of drug diluent or sample solution to be detected and 100 mu L of enzyme-labeled antigen diluted by a certain multiple into each hole; shaking and mixing, incubating at room temperature for 40min, adding 300 μ L of washing solution into each well, washing the plate for 6 times, and spin-drying.
S6, color development: adding 100 μ L of TMB color developing solution into each well, standing at room temperature for 20min, adding 100 μ L of stop solution (10% H) into each well2SO4)。
S7, determination: the absorbance of each well A450nm was measured using an enzyme linked immunosorbent assay.
S8, calculating: establishing a standard curve of methamphetamine by using a four-parameter fitting module of origin8.6, and taking the ratio (B/B) of the absorbance of each standard substance to the absorbance of a zero-concentration standard substance0) As ordinate and logarithm of standard concentration as abscissa, making standard curve, and calculating IC50The value is obtained.
2. Preparing methamphetamine standard solutions of 20ng/mL, 10ng/mL, 4ng/mL, 1ng/mL, 0.25ng/mL and 0.125ng/mL, and establishing a standard curve of the enzyme-linked immunoassay method. Figure 3 is a methamphetamine standard curve. The method is directed to methamphetamine IC501.00ng/mL, linear detection range (IC)20-IC80) 0.22-4.46ng/mL, detection limit (IC)10) 0.13ng/mL, and has no obvious cross connection with analogues such as amphetamine and the like.
EXAMPLE 5 determination of the Cross-reactivity ratio of antibodies
1. Concentration of the best coating antibody obtained in example 4And optimal enzyme-labeled dilution times, and performing ELISA test with structural analogs such as amphetamine, ephedrine, methoxamine, functional analogs such as morphine and ketamine as competitive standard to detect specificity of methamphetamine polyclonal antibody, and half Inhibition Concentration (IC)50) And the values of the cross-reactivity (CR) are given in Table 1.
TABLE 1
Experimental results show that the methamphetamine antibody has no obvious cross with structural or functional analogues of amphetamine, ephedrine, methoxamine, ketamine and morphine. The ephedrine and the methoxamine are main components of common prescription medicaments such as asthma medicaments, and the methamphetamine antibody and the two medicaments are not crossed, so that the methamphetamine antibody has good specificity, the detection result is not easily influenced by the medicaments containing the ephedrine and the methoxamine, and the established enzyme-linked immunoassay method has high accuracy.
The published results of haptens for the specific detection of methamphetamine were retrieved from a review of the literature as follows (table 2). Comparison shows that the antibody obtained by the hapten has higher sensitivity and the hapten is more reasonable in design.
Table 2 partially published methamphetamine hapten structures
Reference [1] Waliluk M, Phenri T, vimolma L.Simultaneous detection of amphetamine, methamphetamine and ephedrine by biotechnology comprehensive enzyme-linked immunological assay [ J ]. Asian Biomedicine,2007,1(2)
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (12)
1. A compound of formula I:
2. a methamphetamine artificial antigen which is a conjugate of a compound of claim 1 and an immunogenic carrier.
3. The methamphetamine artificial antigen of claim 2, having the formula shown in formula II:
4. the methamphetamine artificial antigen of claim 2 or 3, the immunogenic carrier being selected from at least one of keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, immunoglobulins, hormones, and ovalbumin.
5. An antibody obtained by immunizing the methamphetamine artificial antigen according to any one of claims 2 to 4.
6. A methamphetamine assay kit or strip comprising the antibody of claim 5.
7. The kit of claim 6, further comprising one or more of methamphetamine coupled to a signal substance or the compound of claim 1, a solid support, a reaction buffer, a blocking solution, a wash solution, a methamphetamine standard, and a negative control.
8. The kit of claim 7, wherein the antibody is immobilized on the solid support.
9. The kit of claim 8, wherein the solid support is selected from the group consisting of a test tube, an EP tube, a multiwell plate, a microplate well, and a microsphere.
10. The kit according to any one of claims 7 to 9, wherein the signal substance is selected from any one or more of a chromophore, a digoxigenin-labeled probe, an electron-dense substance, colloidal gold, and an enzyme.
11. Use of the antibody of claim 5 or the kit or strip of any one of claims 6 to 10 for detecting methamphetamine.
12. Use according to claim 11, wherein the sample to be detected is blood, urine or hair.
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