CN112724138B - Cocaine hapten, artificial antigen, antibody and application thereof - Google Patents
Cocaine hapten, artificial antigen, antibody and application thereof Download PDFInfo
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- CN112724138B CN112724138B CN202011555671.6A CN202011555671A CN112724138B CN 112724138 B CN112724138 B CN 112724138B CN 202011555671 A CN202011555671 A CN 202011555671A CN 112724138 B CN112724138 B CN 112724138B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
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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 cocaine detection, and particularly relates to a cocaine hapten, an artificial antigen, an antibody and application thereof. The cocaine hapten is a compound shown as a formula I. The cocaine hydroxyl hapten COC-1 is prepared and coupled with different immunogenic carriers to serve as immunogen aiming at cocaine, the prepared polyclonal antibody aiming at cocaine has no obvious cross reaction on common narcotic morphine, ketamine and methamphetamine, the established enzyme-linked immunoassay method can realize specificity detection on cocaine, is convenient and quick, and provides a quick and efficient detection means for detecting cocaine in hair.
Description
Technical Field
The invention relates to the technical field of cocaine detection, and particularly relates to a cocaine hapten, an artificial antigen, an antibody and application thereof.
Background
Cocaine (Cocaine, C)17H21NO4) Commonly known as cocaine, originated in south America, and extracted from coca tree leaves. Cocaine is extracted and purified and then widely used as an anesthetic, a depression medicine and a refreshing beverage, but the abuse of cocaine can cause the dependence of a human body on medicines, chronic poisoning of the human body can be caused by long-term small-dose abuse, and mental symptoms such as anxiety appear, so that cocaine is listed as a controlled medicine by the country and is prohibited from illegal use by the Ming dynasty. The hair is an ideal biological detection material for reflecting whether the suspected person has the abuse history of drugs, the method for quickly screening the cocaine in the hair in high flux is established, the method has important significance for fighting against crimes and suppressing drug abuse, and meanwhile, the requirement for safe physical examination of special high-risk occupational drug involvement is met.
At present, the cocaine detection method mainly adopts a liquid chromatography-mass spectrometry combined method, is accurate and reliable, is often used as a confirmation method, and is not suitable for drug screening of field or large-batch population. The 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. The cocaine enzyme-linked immunosorbent assay (ELISA) method in a research sample 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:
in a second aspect, the invention relates to cocaine artificial antigens which are conjugates of compounds as described above with an immunogenic carrier.
Alternatively, a cocaine artificial antigen as described above having the formula shown in formula II:
optionally, the cocaine 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.
In a third aspect, the invention relates to an antibody, which is obtained by immunizing cocaine artificial antigen as described above.
In a fourth aspect, the invention relates to a cocaine detection kit or strip comprising the antibody described above.
Optionally, the kit as described above, further comprising one or more of cocaine or the compound of formula I coupled with a signal substance, a solid support, a reaction buffer, a blocking solution, a washing solution, a cocaine standard, 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.
In a fifth aspect, the invention also relates to the use of the antibody as described above or the kit or test strip as described above for detecting cocaine.
Alternatively, for use as described above, the sample to be tested is blood, urine or hair.
The invention has the beneficial effects that:
the cocaine hydroxyl hapten COC-1 is prepared aiming at cocaine, different immunogenic carriers are coupled to serve as immunogens, the prepared polyclonal antibody aiming at cocaine does not have obvious cross reaction on common narcotic morphine, ketamine and methamphetamine, the established enzyme-linked immunoassay method can realize specificity detection on cocaine, is convenient and quick, and provides a quick and efficient detection means for detecting cocaine in hair.
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 diagram illustrating the identification of cocaine haptens by mass spectrometry in accordance with 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 cocaine inhibition curve according to 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 cocaine, the hapten which is provided with hydroxyl and is similar to a cocaine structure is preferably synthesized by using ecgonine and p-hydroxybenzoyl chloride, the structural characteristics of functional groups of cocaine are retained to the maximum extent by the hapten, and a cocaine artificial antigen prepared by the hapten can exert the optimal structure-effect advantage, so that an antibody with strong specificity and higher sensitivity is obtained.
There are few published studies on cocaine haptens, most of which extend the carbon chain arm at the azomethine end of cocaine. Compared with the cocaine structure, the cocaine hapten (COC-1) introduces hydroxyl on a benzene ring, so that characteristic groups of cocaine are exposed in animal immune response, and an animal body is promoted to generate high-efficiency antibodies.
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 cocaine artificial antigen which is a conjugate of the compound and an immunogenic carrier.
In some embodiments, the cocaine 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 substances 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 the host. The polyaminopolysaccharides may be prepared from the polysaccharides using any conventional method known for such preparation.
A variety of 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 include 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 antibodies raised against cocaine artificial antigens as described above.
The term "antibody" refers to a specific protein capable of binding to an antigen or a part thereof (which according to the invention is capable of binding to an antipsychotic drug or a metabolite thereof). 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 cocaine artificial antigen is initially injected into various breeds of any mammal (e.g., mouse, rat, rabbit, sheep, and goat). The cocaine artificial antigen can also be combined with immune adjuvant (such as Freund's complete adjuvant and Freund's incomplete adjuvant) to inject immunogen into 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 produced 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 cocaine detection kit containing the antibody.
In some embodiments, the kit further comprises one or more of cocaine or a compound of formula I coupled to a signal substance, a solid support, a reaction buffer, a blocking solution, a wash solution, a cocaine standard, and a negative control.
In some embodiments, the kit further comprises a chromogenic reagent for the signal substance (e.g., if the signal substance is horseradish peroxidase, then the chromogenic reagent is a TMB chromogenic solution)
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 (elemental metal or alloy), a nonmetal, or a composite of a metal and a nonmetal. Metals such as iron, alnico, etc.; 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 variations, and contact variation angles) or physical methods (e.g., atomic force 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 cocaine detection.
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 specific detection of cocaine residue in hair, and the method is used for the IC of cocaine50The linear detection range is between 0.39 and 11.61ng/mL and the detection limit is 0.11ng/mL, which is 2.13 ng/mL. 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 for cocaine. The method is stable and reliable, has low cost, and has important significance for research and development of cocaine rapid detection kits and colloidal gold test strips.
Embodiments of the present invention will be described in detail with reference to examples.
EXAMPLE 1 preparation of haptens
Adding 50.0mg of ecgonine (CAS: 481-37-8) and 84.5mg of p-hydroxybenzoyl chloride into 15ml of tetrahydrofuran, adding 63.4mg of nickel into the mixed solution, connecting the mixed solution with a water separator, heating in a water bath at 70 ℃ for reacting for 4 hours, and after the reaction is finished, extracting the product mixed solution with ethyl acetate for 3 times, wherein the volume ratio of the extracted mixed solution to the ethyl acetate is 1: 2; and (3) drying the extract liquid in a spinning way, purifying the extract liquid through a silica gel column (methanol: chloroform, 1: 15, V/V), and evaporating the extract liquid to dryness to obtain powdery mildew, namely hapten COC-1, wherein a figure 1 is a COC-1 mass spectrum identification chart, and the hapten is successfully synthesized.
EXAMPLE 2 preparation of Artificial vector
1. Preparation of immunogen COC-1-KLH
20.0mg of the COC-1 hapten was dissolved in 0.20mL of dimethyl sulfoxide and designated as solution A. Dissolving 36.9mg of KLH in 6mL of 0.1mol/L acetic acid buffer solution, marking as solution B, dripping the solution A into the solution B under stirring, adding 300 mu L of formaldehyde solution (31%), reacting for 12 hours at 30 ℃ in a dark place, centrifuging after the reaction is finished, taking supernatant, dialyzing for 3 days by using PBS (phosphate buffer solution) at 4 ℃ to obtain cocaine artificial immunogen, subpackaging the cocaine artificial immunogen in 1mL of centrifuge tubes at the concentration of 1mg/mL, and freezing and storing the cocaine artificial immunogen in a refrigerator at-20 ℃ for later use.
2. Preparation of enzyme-labeled antigen COC-1-HRP
20.0mg of the COC-1 hapten was dissolved in 0.20mL of dimethyl sulfoxide and designated as solution A. Dissolving 27.0mg of HRP in 6mL of 0.1mol/L acetic acid buffer solution to be marked as solution B, dripping the solution A into the solution B under stirring, adding 300 mu L of formaldehyde solution (31%), reacting for 12 hours at 30 ℃ in a dark place, centrifuging after the reaction is finished, taking supernatant, dialyzing for 3 days by PBS solution at 4 ℃ to obtain cocaine enzyme-labeled antigen, subpackaging in 1mL of centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use.
3. Characterization of cocaine drug immunogens and enzyme-labeled antigens
(1) And the immunogen and the enzyme-labeled antigen are identified by adopting an ultraviolet scanning method, measuring the ultraviolet absorption spectra of the immunogen and the coating antigen within the wavelength range of 200-300 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 preparation of cocaine polyclonal antibodies
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 Freund incomplete adjuvant is used for emulsification; 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 (4) 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 cocaine standard in appropriate amount of methanol to obtain 1.0mg/mL standard solution, and storing at 4 deg.C. 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 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 hybrid 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 then 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 a method for enzyme-linked immunoassay of cocaine in hair
1. Enzyme-linked immunosorbent assay (ELISA) reactions specifically comprise the following steps:
s1, coating: the cocaine antibody was diluted to the appropriate concentration with carbonate buffer, added to the wells of an enzyme-labeled plate at 100. mu.L/well, and incubated overnight in a 37 ℃ water bath.
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 development solution into each well, standing at room temperature for 20min, and 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: the ratio (B/B) of the absorbance of each standard substance to the absorbance of the standard substance with zero concentration0) On the ordinate, the logarithm of the concentration of the standard is plotted on the abscissa, and the IC of the inhibition curve is calculated using a four-parameter fitting model from origin8.650The value is obtained.
2. Preparing cocaine standard solutions of 100ng/mL, 25ng/mL, 6.25ng/mL, 2.08ng/mL, 0.69ng/mL and 0.08ng/mL, and establishing a standard curve of the enzyme-linked immunoassay method. Figure 3 is a cocaine inhibition curve. The method is directed to cocaine IC50The linear detection range is 0.39-11.61ng/mL, and the detection limit is 0.11 ng/mL.
EXAMPLE 5 determination of the Cross-reactivity ratio of antibodies
1. According to the optimal concentration of the coating antibody and the optimal enzyme-labeled dilution multiple obtained in the example 4, common drugs morphine, ketamine and methamphetamine are used as competitive standards to carry out ELISA experiments to detect the specificity of the cocaine polyclonal antibody, and the half Inhibitory Concentration (IC) of the cocaine polyclonal antibody50) And cross-reactivity (CR) values are listed in table 1.
TABLE 1
Experimental results show that the cocaine antibody has no obvious cross with common drugs morphine, ketamine and methamphetamine, and the cocaine antibody has good specificity and the established enzyme-linked immunoassay method has high specificity.
Few articles and patents are published on cocaine hapten design at present, the cocaine hapten (COC-1) is synthesized by ecgonine and p-hydroxybenzoyl chloride, and in the actual immune result, the obtained cocaine antibody IC50It was 2.13 ng/mL. The reference literature shows that the antibody obtained by the hapten of the patent has higher sensitivity and more reasonable design of the hapten (Table 2).
Table 2 published structures of cocaine haptens
Reference to the literature
[1] Zheng Shunjing, Zheng Chibushi, Liujing, anti-cocaine monoclonal antibody, cell strain secreting the antibody and preparation method (CN 107988168A), 2017.
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 specific and detailed, but not to be understood 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 (9)
1. A cocaine artificial antigen having a chemical formula shown in formula II:
2. the cocaine artificial antigen of claim 1, the immunogenic carrier selected from at least one of keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, lactoferrin, horseradish peroxidase, thyroglobulin, an immunoglobulin, a hormone, and an ovalbumin.
3. An antibody immunized with the cocaine artificial antigen of claim 1 or 2.
4. A cocaine detection kit or strip comprising the antibody of claim 3.
5. The kit of claim 4, further comprising one or more of cocaine or a compound of formula I coupled to a signal substance, a solid support, a reaction buffer, a blocking solution, a wash solution, a cocaine standard, and a negative control;
a compound of formula I:
the signal substance is selected from any one or more of chromophore, digoxin labeled probe, electron dense substance, colloidal gold and enzyme.
6. The kit of claim 5, wherein said antibody is immobilized coated on said solid support.
7. The kit of claim 6, wherein the solid support is selected from the group consisting of test tubes, EP tubes, multiwell plates, microplate wells, and microspheres.
8. The kit of claim 7, wherein the microspheres have a diameter of 10nm to 1 mm.
9. The kit of claim 7, wherein the multi-well plate is an elisa plate.
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