阅读说明：本技术 氯胺酮半抗原、人工抗原、抗体及其应用 (Ketamine hapten, artificial antigen, antibody and application thereof ) 是由 刘晓云 黄健欣 袁强 于 2020-12-18 设计创作，主要内容包括：本发明涉及氯胺酮检测技术领域,具体而言,涉及一种氯胺酮半抗原、人工抗原、抗体及其应用。氯胺酮半抗原,其为式I所示的化合物。本发明针对氯胺酮,制备通过氯胺酮分子苯环上衍生丁酸手臂的羧基型半抗原KET-1作为免疫原和酶标抗原,制备的针对氯胺酮的多克隆抗体,对常见毒品甲基苯丙胺、吗啡、可卡因、四氢大麻酚无明显交叉反应,建立的酶联免疫检测方法可以实现对氯胺酮的特异性检测,方便快捷,为检测毛发中氯胺酮提供了快速高效的检测手段。(The invention relates to the technical field of ketamine detection, and in particular relates to a ketamine hapten, an artificial antigen, an antibody and application thereof. Ketamine hapten which is a compound of formula I. The invention aims at ketamine, the carboxyl hapten KET-1 of butyric acid arm derived from the benzene ring of ketamine molecule is prepared as immunogen and enzyme-labeled antigen, the prepared polyclonal antibody aiming at ketamine has no obvious cross reaction to common drugs of methamphetamine, morphine, cocaine and tetrahydrocannabinol, the established enzyme-linked immunoassay method can realize the specificity detection of ketamine, is convenient and quick, and provides a quick and efficient detection means for detecting ketamine in hair.)

1. A compound of formula I:

2. a ketamine artificial antigen which is a conjugate of the compound of claim 1 and an immunogenic carrier.

3. The ketamine artificial antigen of claim 2, having the formula shown in formula II:

4. the ketamine artificial antigen of claim 2 or 3, wherein the immunogenic carrier is 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 immunized with the ketamine artificial antigen of any one of claims 2-4.

6. A ketamine assay kit or strip comprising the antibody of claim 5.

7. The kit of claim 6, further comprising ketamine conjugated to a signal substance or the compound of claim 1, a solid support, a reaction buffer, a blocking solution, a wash solution, a ketamine standard, and one or more of 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 the detection of ketamine.

12. Use according to claim 11, wherein the sample to be detected is blood, urine or hair.

Technical Field

The invention relates to the technical field of ketamine detection, and in particular relates to a ketamine hapten, an artificial antigen, an antibody and application thereof.

Background

Ketamine (C)₁₃H₁₆ClNO, Ketamine) is white powder, and is generally used as an anesthetic in medical clinics. After eating, the food can generate consciousness and feeling separation states to cause psychopathic symptoms such as confusion, depression after overexcitation and the like, and the state puts the food into the management and control of narcotics, and the state prohibits illegal use. The drug taking ketamine as the main component is commonly called K powder, and excessive smoking can easily cause bladder atrophy and even cause respiratory arrest. After the ketamine is taken, drug residues can be accumulated in the hair for a long time, and the 3cm of hair roots close to the scalp can reflect the drug taking history of 6 months, so that the ideal biological material for hair material testing judicial identification is provided. The establishment of the rapid high-throughput screening method for ketamine in 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.

At present, a method for detecting ketamine mainly adopts liquid chromatography-mass spectrometry, the method is accurate and reliable, and is often used as a confirmation method, but the method has the defects of expensive equipment, professional requirement, long time consumption and the like, and is not suitable for large-scale screening. 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. An enzyme-linked immunosorbent assay (ELISA) method for researching ketamine 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 a ketamine artificial antigen which is a conjugate of a compound as described above and an immunogenic carrier.

Alternatively, a ketamine artificial antigen as described above having the formula shown in formula II:

optionally, the ketamine 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, immunoglobulins, hormones, and ovalbumin.

According to a third aspect of the invention, it relates to an antibody, which is immunized with the ketamine artificial antigen as described above.

According to a fourth aspect of the invention, it relates to a ketamine assay kit or strip containing an antibody as described above.

Optionally, the kit as described above, further comprising one or more of ketamine coupled to a signal substance or a compound of formula I, a solid support, a reaction buffer, a blocking solution, a wash solution, a ketamine 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 microwell plate, 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 invention, it also relates to the use of an antibody as described above or a kit or test strip as described above for the detection of ketamine.

Alternatively, for use as described above, the sample to be tested is blood, urine or hair.

The invention has the beneficial effects that:

aiming at ketamine, the invention prepares carboxyl hapten KET-1 of a butyric acid arm derived from a benzene ring of a ketamine molecule and couples different immunogenic carriers as immunogen and enzyme-labeled antigen, the prepared polyclonal antibody aiming at the ketamine has no obvious cross reaction on common drugs of methamphetamine, morphine, cocaine and tetrahydrocannabinol, the established enzyme-linked immunoassay method can realize the specific detection of the ketamine, is convenient and quick, and provides a quick and efficient detection means for detecting the ketamine 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 chart of KET-1 mass spectrometric identification 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 for ELISA detection of ketamine 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. Indeed, 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 ketamine, the hapten furthest retains the structural characteristics of a functional group of the ketamine, and the ketamine 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.

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 ketamine artificial antigen which is a conjugate of the compound and an immunogenic carrier.

In some embodiments, the ketamine 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 materials 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, which are obtained by immunizing the ketamine artificial antigen as described above.

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, immunogens comprising ketamine artificial antigens are initially injected into any of a variety of mammals (e.g., mice, rats, rabbits, sheep, and goats). The ketamine artificial antigen can also be combined with an immunological adjuvant (such as Freund's complete adjuvant and Freund's incomplete adjuvant) to inject the immunogen into an animal host; more preferably, the injection is performed 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 by affinity chromatography, for example, 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 or have the potential to secrete monoclonal antibodies, 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 ketamine detection kit containing the antibody.

In some embodiments, the kit further comprises one or more of ketamine coupled to a signal substance or a compound of formula I, a solid support, a reaction buffer, a blocking solution, a wash solution, a ketamine 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 spherical, nearly spherical, cubic, polyhedral or irregularly shaped. 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 nonmetal 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 use of an antibody as described above or a kit as described above for the detection of ketamine.

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 ketamine residue in hair, and the IC of the method for ketamine₅₀1.41ng/mL, linear detection range (IC)₂₀-IC₈₀) 0.07-29.24 ng/mL, detection limit (IC)₂₀) 0.07ng/mL, and has no cross effect with common drugs such as methamphetamine, morphine, cocaine, and tetrahydrocannabinol. 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 immune detection method of ketamine. The method is stable and reliable, has low cost, and has important significance for the research and development of ketamine 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

Under the protection of nitrogen, adding ketamine hydrochloride 50mg and 4-bromobutyric acid 36.5mg into 15mL of acetonitrile, sequentially adding KI 24.2mg, catalyst 18-crown-64.8 mg and potassium carbonate 30.2mg into the mixed solution, carrying out reflux reaction in a water bath at 80 ℃ for 24 hours, removing the solvent under reduced pressure after the reaction is finished, adding ice water 10mL, stirring and dissolving, extracting the product mixed solution for 3 times by using ethyl acetate, wherein the volume ratio of the mixed solution to the ethyl acetate in each extraction is 1: 2; the extract was spin-dried and purified by silica gel column (methanol: chloroform, 1/15, V/V) to obtain a pale yellow powder as hapten KET-1, FIG. 1 is a figure of mass spectrometric identification of KET-1.

EXAMPLE 2 preparation of Artificial vector

1. Preparation of immunogen KET-1-KLH

15.0mg of KET-1 hapten is dissolved in 0.30mL of N, N-dimethylformamide, 14.34 mg of N, N-dicyclohexylcarbodiimide and 8.00mg of N-hydroxysuccinimide are added, the mixture is stirred at 4 ℃ for reaction overnight, and after centrifugation, the supernatant is taken and recorded as solution A. Dissolving 34.74mg of KLH in 6mL of 0.01mol/L PBS (phosphate buffer solution) to obtain solution B, dripping the solution A into the solution B while stirring, reacting for 12 hours in a dark place at 4 ℃, centrifuging after the reaction is finished, taking supernatant, dialyzing by using the PBS solution at 4 ℃ for 3 days to obtain the ketamine artificial immunogen, subpackaging the ketamine artificial immunogen into 1mL of centrifuge tubes at the concentration of 1mg/mL, and freezing and storing in a refrigerator at-20 ℃ for later use.

2. Preparation of enzyme-labeled antigen KET-1-HRP

15.0mg of KET-1 hapten is dissolved in 0.30mL of N, N-dimethylformamide, 14.34 mg of N, N-dicyclohexylcarbodiimide and 8.00mg of N-hydroxysuccinimide are added, the mixture is stirred at 4 ℃ for reaction overnight, and after centrifugation, the supernatant is taken and recorded as solution A. Dissolving 25.47mg of HRP in 6mL of 0.01mol/L PBS (phosphate buffer solution), recording 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 ketamine standard antigen, subpackaging in 1mL of centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use.

3. Characterization of ketamine drug 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 180-300 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 result of ultraviolet spectrum scanning can prove that the immunogen and the enzyme-labeled antigen are successfully coupled.

EXAMPLE 3 preparation of polyclonal antibodies to Ketamine drugs

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 was collected, antiserum was collected by centrifugation, the antibody concentration was detected by nanodrop, and the samples were stored at-20 ℃ for further use.

(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 ketamine 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 performing 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 a method for the enzyme-linked immunoassay of ketamine in hair

1. The enzyme-linked immunosorbent assay (ELISA) reaction specifically comprises the following steps:

s1, coating: the ketamine antibody was diluted to the appropriate concentration with carbonate buffer, added to the wells of the microplate, 100 μ 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 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: the ratio (B/B) of the absorbance of each standard substance to the absorbance of the standard substance with zero concentration₀) For ordinate, the logarithm of the concentration of the standard is plotted on abscissa, and the IC of the inhibition curve is calculated using a four-parameter fitting model from origin8.6₅₀The value is obtained.

2. Preparing ketamine standard solutions of 30ng/mL, 20ng/mL, 10ng/mL, 4ng/mL, 1ng/mL, 0.5ng/mL and 0.25ng/mL, and establishing a standard curve of the enzyme-linked immunoassay method. FIG. 3 is a standard curve for chloraminoketone. The method is directed to ketamine IC₅₀The linear detection range is 0.07 ng/mL-29.24 ng/mL and 1.41ng/mLThe detection limit is 0.07ng/mL, and the detection limit does not intersect with common drugs of ketamine, morphine, cocaine and tetrahydrocannabinol.

EXAMPLE 5 determination of the Cross-reactivity ratio of antibodies

1. ELISA experiments were performed according to the optimal concentration of the coating antibody and the optimal dilution factor of the enzyme label obtained in example 4, using the common drugs ketamine, morphine, cocaine and tetrahydrocannabinol as competitive standards, to detect the specificity of the polyclonal antibody to chloramine, the median Inhibitory Concentration (IC)₅₀) And the values of the cross-reactivity (CR) are given in Table 1.

TABLE 1

Experimental results show that the ketamine antibody has no cross with common drugs of ketamine, morphine, cocaine and tetrahydrocannabinol, and the ketamine antibody has good specificity and the established enzyme-linked immunoassay method has high specificity.

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.