CN112480193A - Artificial hapten, artificial antigen and polyclonal antibody of opium narcotics and application thereof - Google Patents
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Abstract
The invention provides an opium drug artificial hapten, an artificial antigen, a polyclonal antibody and application thereof, wherein the opium drug artificial hapten is a hydroxyl-derived D-glucuronic acid arm on a morphine molecule phenol ring, the opium drug artificial hapten is coupled with carrier protein to obtain an opium drug artificial antigen, and the opium drug artificial hapten is coupled with horseradish peroxidase to obtain an enzyme-labeled antigen. The method is used for qualitative and quantitative detection of opioid drugs, has the remarkable advantages of short time consumption, high sensitivity, good specificity and the like, and can be used for quickly detecting the residual opioid drugs in the hair.
Description
Technical Field
The invention belongs to the technical field of drug detection, and relates to an opioid drug artificial hapten, an artificial antigen, a polyclonal antibody and application thereof.
Background
The opioid narcotics are usually morphine, codeine and monoacetylmorphine, which are the main components of heroin. Morphine (Morphine, C)17H19NO3) Codeine (C)18H21NO3) Is an opioid receptor agonist and is the most predominant alkaloid in opiates. Monoacetylmorphine (Morphine 6-acetate, C)19H21NO4) Is obtained by formylating crude morphine base or morphine salt in opium, and is the main component of hypoheroin. According to the Chinese drug situation report of 2019, the types of drugs are the highest among drug addicts, such as methamphetamine, heroin and K powder. Heroin is the most widely abused drug in the world today, has high addiction rate and strong dependence, and is difficult to be withdrawn. Excessive inhalation of opioid drugs can easily cause palpitation, and the drugs have great damage to liver and kidney and even die after being inhaled into human bodies, so the drugs are listed in the drug management and regulated by the nation, and are forbidden to be illegally used by the nation. After morphine, codeine and monoacetylmorphine enter a human body, accumulation can be formed in biological detection of blood, urine, hair and the like, but drug residues in body fluid are rapidly metabolized, and the detection cannot be carried out after 2-4 days generally. Compared with body fluid detection material, the hair detection material has the advantages of easy sampling, civilization, difficult adulteration, stable detection material, easy storage, convenient carrying, long detection period and the like. It is generally considered that the 3cm root close to the scalp reflects the 6 months history of drug absorption, so the hair test material has become the most commonly used biological test material in judicial identification. Therefore, the establishment of the rapid high-throughput screening method for opium in hair has important significance for fighting against crimes and suppressing drug abuse, and simultaneously meets the requirements of special high-risk occupational drug-involved safety physical examination.
At present, the method for detecting opioid drugs is mainly liquid chromatography-mass spectrometry, is accurate and reliable, is often used as a confirmation method, and has the defects of expensive equipment, professional requirement, low sample flux and the like. The immunoassay technology is an analysis method based on antigen-antibody specific reaction, has the characteristics of sensitivity, rapidness, high flux 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 opioid narcotics in hair has very important economic and social significance for quickly screening and detecting narcotics in large-scale samples.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an opioid narcotic artificial hapten, an artificial antigen, a polyclonal antibody and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the present invention provides an opioid drug artificial hapten having the following structure:
the opioid drug artificial hapten is a hydroxyl-derived D-glucuronic acid arm on a phenol ring of a morphine molecule, and is abbreviated as MOP-1.
In another aspect, the present invention provides a method for preparing the opioid drug artificial hapten, wherein the method comprises the following steps: morphine reacts with D-glucuronic acid to obtain the opioid drug artificial hapten.
Preferably, the molar ratio of morphine to D-glucuronic acid is 1: 1.5-3, such as 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, 1:2.8 or 1:3, preferably 1:2.
Preferably, the reaction is carried out under weakly acidic conditions.
Preferably, the reaction is carried out in the presence of a catalyst, preferably anhydrous zinc chloride.
Preferably, the molar ratio of the catalyst to morphine is 3 to 6:1, such as 3:1, 3.5:1, 3.8:1, 4:1, 4.5:1, 4.8:1, 5:1, 5.5:1, 5.8:1 or 6:1, preferably 4: 1.
Preferably, the solvent of the reaction is anhydrous tetrahydrofuran.
Preferably, the temperature of the reaction is 30 to 70 ℃ (e.g., 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 60 ℃ or 70 ℃), preferably 50 ℃; the reaction time is 3 to 7 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, or 7 hours.
After the reaction is finished, extracting the product mixed liquor with ethyl acetate for 3 times, preferably, the volume ratio of the mixed liquor to the ethyl acetate in each extraction is 1: 2; and (3) drying the extract liquor in a spinning way, purifying the extract liquor by using a reverse phase liquid chromatography, and evaporating the extract liquor to dryness to obtain light yellow powder, namely the artificial hapten.
In another aspect, the present invention provides an opioid drug artificial antigen, which is a conjugate of the opioid drug artificial hapten and a carrier protein as described above.
In the present invention, the opioid narcotic artificial antigen can be used as an artificial immunogen.
In the present invention, the structure of the opioid drug artificial antigen may be represented as:
Preferably, the carrier protein is any one of Keyhole Limpet Hemocyanin (KLH), Bovine Serum Albumin (BSA), Lactoferrin (LF), horseradish peroxidase (HRP), or Ovalbumin (OVA).
In another aspect, the present invention provides a method for preparing the opioid drug artificial antigen, wherein the method comprises: dissolving the opioid drug artificial hapten in an organic solvent, adding an activating agent, then adding a buffer solution dissolved with carrier protein, and reacting to obtain the opioid drug artificial antigen.
Preferably, the organic solvent is N, N-Dimethylformamide (DMF).
Preferably, the activator is a mixture of N, N-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS).
Preferably, the molar ratio of the opioid drug artificial hapten to the carrier protein is (25-100): 1, e.g. 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 70:1, 80:1, 90:1 or 100:1, preferably 80: 1.
Preferably, the molar ratio of the opioid drug artificial hapten to N, N-Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) is 1 (1-2), such as 1:1:1, 1:1.1:1, 1:1.2:1, 1:1.5:1, 1:1.8:1, 1:2:1, 1:1:1.2, 1:1:1.5, 1:1:1.8, 1:1:2, 1:2:1, 1:2:1.3, 1:2:1.5, 1:2:1.8, 1:2:2, etc., preferably 1:1.5: 1.5.
Preferably, the buffer solution is a PBS solution.
Preferably, the concentration of the PBS solution is 0.005-0.015 mol/L, such as 0.005mol/L, 0.008mol/L, 0.01mol/L, 0.012mol/L, 0.014mol/L or 0.015mol/L, preferably 0.01 mol/L.
Preferably, the temperature of the reaction is 0-10 ℃ (e.g., 0 ℃, 3 ℃, 5 ℃, 8 ℃, 10 ℃), preferably 4 ℃; the reaction time is 10 to 20 hours (for example, 10 hours, 13 hours, 15 hours, 17 hours, 20 hours), preferably 16 hours.
In the preparation process of the opioid drug artificial antigen, after the reaction is finished, the opioid drug artificial antigen is obtained by dialysis with PBS buffer solution at 4 ℃.
In another aspect, the present invention provides a polyclonal antibody, which is obtained by injecting the opioid drug artificial antigen as described above into an experimental animal and immunizing the animal for a plurality of times.
In the invention, the polyclonal antibody can be used as a coating antigen in opioid drug detection.
In another aspect, the invention provides an opioid drug enzyme-labeled antigen, which is a conjugate of the opioid drug artificial hapten and horseradish peroxidase.
In another aspect, the present invention provides an immunological detection kit for opioid drugs, which includes the polyclonal antibody and the enzyme-labeled antigen for opioid drugs.
The immunological detection kit can be used for qualitative and quantitative detection of opioid drugs, has the remarkable advantages of short time consumption, high sensitivity, good specificity and the like, and can be used for quickly detecting the residual opioid drugs in the hair.
In another aspect, the present invention provides the use of the above opioid drug artificial antigen or the above polyclonal antibody or the above opioid drug enzyme-labeled antigen or the above opioid drug immunological detection kit in the opioid drug immunoassay.
Preferably, the immunoassay for opioid narcotics is an immunoassay for opioid narcotics remaining in the hair.
On the other hand, the invention provides an enzyme-linked immunoassay method for opioid narcotics in hair, which comprises the following steps: coating the polyclonal antibody on a microplate; adding the hair sample solution to be detected and the enzyme-labeled antigen of the opioid narcotic drug into the micropores coated with the polyclonal antibody, and determining the content of the opioid narcotic drug in the sample to be detected by adopting an enzyme-linked immunosorbent assay method.
In the invention, the preparation method of the hair sample solution to be tested comprises the following steps: adding 20-25 mg of hair to be detected, 10-25 zirconium beads with the width of 2mm, 0.5-1.5 mL of PBS (pH 6.0-9.0) or sodium borate buffer solution (pH 8.0-10.0) into a grinding tube, and grinding in a vibration grinding instrument for 3-5min to obtain an upper layer solution, namely a sample solution to be detected.
In the invention, specifically, the enzyme-linked immunoassay method for opioid narcotics in hair comprises the following steps:
s1, coating: diluting the opium drug antibody by certain times with carbonic acid buffer solution, making blank control group, incubating at 37 deg.C overnight with 100 μ L of each well;
s2, washing: pouring out liquid in the holes, washing for 2 times by deionized water, and spin-drying;
s3, sealing: adding 120 μ L of sealing liquid into each hole, sealing at 37 deg.C for 3 hr, drying, and placing in oven at 37 deg.C for 1 hr;
s4, sample adding and incubation: the standard morphine was dissolved in methanol to make a 1.0mg/mL solution. Firstly, diluting the sample solution into a standard solution with a certain concentration by using 0.01mol/L PBS buffer solution, and totaling 7 concentration gradients; adding 20 mu L of standard solution diluted in gradient or 20 mu L of sample solution to be detected into each hole of the ELISA plate, oscillating and uniformly mixing with 100 mu L of enzyme-labeled antigen with specific concentration, reacting at normal temperature for 40min, washing for 6 times, and spin-drying;
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 10% H into each well2SO4Terminating the reaction;
s6, reading measurement: measuring the light absorption value at the wavelength of 450 nm; selecting antiserum dilution times with absorbance values within the range of 1.0-1.5 as antiserum titer, wherein the antiserum effect is obtained by the inhibition rate;
s7, calculating: establishing standard curve of morphine by using four-parameter fitting module of origin8.6, and taking ratio (B/B) of absorbance of each standard substance to absorbance of a zero-concentration standard substance0) As ordinate and logarithm of concentration of standard substance as abscissa, making standard curve, and calculating IC50A value; and (3) bringing the absorbance value of the sample to be detected into a standard curve, and carrying out qualitative and quantitative detection on the opium drugs in the sample to be detected.
Compared with the prior art, the invention has the following beneficial effects:
(1) aiming at opioid narcotics, morphine is used as a synthetic raw material, and a compound with a structure similar to that of glucoside in an animal body is introduced, so that hapten coupling protein ends are not easy to respond to the immune response of the organism; according to the design idea of the hapten, morphine and D-glucuronic acid are subjected to condensation reaction to synthesize the hapten MOP-1; on one hand, the morphine molecular structure has the characteristic parent structure of the opioid, and the morphine molecule is taken as a hapten synthetic material, so that an organism is more easily caused to generate antibodies aiming at the opioid; on the other hand, the addition of the glucoside structure increases the distance between the small molecular part of the morphine in the hapten and the carrier protein, so that the small molecular part of the morphine in the hapten can be fully exposed. In the actual immune result, the obtained opioid antibody has high sensitivity and can identify various opioid drugs;
(2) the artificial antigen obtained by coupling the opioid drug artificial hapten and the carrier protein can be used as immunogen, and the opioid drug artificial hapten is coupled with horseradish peroxidase to be used as enzyme-labeled antigen. The method can be used for qualitative and quantitative detection of opioid drugs, has the remarkable advantages of short time consumption, high sensitivity, good specificity and the like, and can be used for quickly detecting the residual opioid drugs in the hair.
(3) Aiming at the polyclonal antibody of the opium narcotic, the invention has no obvious cross reaction on structural analogues of papaverine, common narcotic methamphetamine and ketamine, the established enzyme-linked immunoassay method can realize the specific detection of the opium narcotic, is convenient and quick, and provides a quick and efficient detection means for detecting the opium narcotic in hair.
(4) The antibody and the enzyme-linked immunoassay method can be used for the specificity detection of opioid drug residues in hair, and the IC of the method for morphine500.13ng/mL, linear detection range (IC)20-IC80) 0.04-0.48ng/mL, detection limit (IC)10) The concentration of the active compound is 0.03ng/mL, and the crossing rate of the active compound with codeine and monoacetylmorphine is 115.38 percent and 92.9 percent respectively. The antibody has the obvious advantages of high sensitivity, good accuracy, simple and quick sample pretreatment method and the like, so the antigen and the antibody provided by the invention can be used for establishing an enzyme-linked immunoassay method for opioid drugs. The method is stable and reliable, has low cost, and has important significance for the research and development of opioid drug rapid detection kits and colloidal gold test strips.
Drawings
FIG. 1 is a mass spectrum of the opioid drug artificial hapten prepared in example 1;
FIG. 2 is a diagram showing ultraviolet absorption spectra of BSA, MOP-1-BSA, HRP, and MOP-1-HRP;
figure 3 is a morphine standard curve obtained in the enzyme-linked immunoassay of example 4.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
EXAMPLE 1 preparation of haptens
Adding 580mg of morphine and 780mg of D-glucuronic acid into 15mL of tetrahydrofuran, adding 1090mg of anhydrous zinc chloride into the mixed solution, connecting the mixed solution with a water separator, heating the mixed solution in a water bath at 50 ℃ for reaction 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 by reverse phase liquid chromatography, and evaporating the extract liquid to dryness to obtain light yellow powder, namely hapten MOP-1, wherein the figure 1 is a MOP-1 mass spectrum identification figure, and the hapten is successfully synthesized.
EXAMPLE 2 preparation of Artificial vector
1. Preparation of immunogen MOP-1-BSA
20.0mg of MOP-1 hapten is dissolved in 0.30mL of N, N-dimethylformamide, 13.41mg of N, N-dicyclohexylcarbodiimide and 7.48mg 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 35.97mg 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 for 3 days by using the PBS solution at 4 ℃ to obtain the morphine artificial immunogen, subpackaging the morphine artificial immunogen into 1mL centrifuge tubes at the concentration of 1mg/mL, and freezing in a refrigerator at-20 ℃ for later use.
2. Preparation of enzyme-labeled antigen MOP-1-HRP
20.0mg of MOP-1 hapten is dissolved in 0.30mL of N, N-dimethylformamide, 13.41mg of N, N-dicyclohexylcarbodiimide and 7.48mg 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 24.38mg 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 morphinase labeled antigen, subpackaging in 1mL of centrifuge tubes, and freezing in a refrigerator at-20 ℃ for later use.
3. Characterization of opioid 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 180-350 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 preparation of polyclonal antibody to opioid 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 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 morphine standard substance in appropriate amount of methanol to obtain 1.0mg/mL standard substance 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 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 opioid drugs in hair
1. The enzyme-linked immunosorbent assay (ELISA) reaction specifically comprises the following steps:
s1, coating: the opioid drug antibody is diluted to a proper concentration by carbonic acid buffer solution, added into the well of the enzyme-labeled plate, and added into the water bath box at 37 ℃ overnight at a concentration of 100 mu L/well.
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, and developing at room temperature for 20minAdd 100. mu.L of stop solution (10% H) to each well2SO4)。
S7, determination: the absorbance of each well A450nm was measured using an enzyme linked immunosorbent assay.
S8, calculating: IC of inhibition curves calculated using the four parameter fitting Module of origin8.650The value is obtained.
2. Preparing morphine standard solution with 4ng/mL, 1ng/mL, 0.5ng/mL, 0.25ng/mL, 0.125ng/mL, 0.063ng/mL and 0.031ng/mL, and establishing a standard curve of the enzyme-linked immunoassay method. 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 substance was plotted on the abscissa, and a standard curve was prepared, and fig. 3 is a graph showing the resulting morphine inhibition curve. The method is directed to morphine IC50The linear detection range is 0.04-0.48ng/mL, the detection limit is 0.03ng/mL, and the crossing rates with codeine and monoacetylmorphine are 115.4% and 92.9%, respectively.
EXAMPLE 5 determination of the Cross-reactivity ratio of antibodies
1. ELISA test was performed according to the optimal concentration of coating antibody and the optimal enzyme-labeled dilution factor obtained in example 4, using monoacetylmorphine, papaverine, ketamine, methamphetamine structural analogue or functional analogue as competitive standard, to detect the specificity of polyclonal antibody of opioid drug, and its half Inhibitory Concentration (IC)50) And the values of the cross-reactivity (CR) are given in Table 1.
TABLE 1
The experimental result shows that the opioid antibody has no cross with papaverine, ketamine and methamphetamine which are structural or functional analogues, and the opioid antibody has good specificity and the established enzyme-linked immunoassay method has high specificity.
The opioid hapten (MOP-1) is synthesized from D-glucuronic acid and morphine, and the D-glucuronic acid has a structure similar to that of endogenous glucoside in an animal body, so that the end connected with a carrier protein is not easy to cause immune reaction in the animal body; on the other hand, the addition of the glucoside structure increases the distance between the small molecular part of the morphine in the hapten and the carrier protein, so that the small molecular part of the morphine in the hapten can be fully exposed. In the actual immunization results, the obtained opioid antibody IC50It was 0.13 ng/mL. The published hapten results for opioid assays were retrieved by reference to 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 opioid hapten structures
The applicant states that the present invention is illustrated by the above examples to show the opioid drug artificial hapten, artificial antigen, polyclonal antibody and application thereof, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the examples. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (10)
1. An opioid drug artificial hapten, wherein the opioid drug artificial hapten has the following structure:
2. the method of claim 1, wherein the method comprises: morphine reacts with D-glucuronic acid to obtain the opioid drug artificial hapten.
3. The preparation method according to claim 2, wherein the molar ratio of morphine to D-glucuronic acid is 1: 1.5-3, preferably 1: 2;
preferably, the reaction is carried out under weakly acidic conditions;
preferably, the reaction is carried out in the presence of a catalyst, preferably anhydrous zinc chloride;
preferably, the molar ratio of the catalyst to morphine is 3-6: 1, preferably 4: 1;
preferably, the solvent of the reaction is anhydrous tetrahydrofuran;
preferably, the reaction temperature is 30-70 ℃, preferably 50 ℃; the reaction time is 3-7 hours.
4. An opioid drug artificial antigen, which is a conjugate of the opioid drug artificial hapten as claimed in claim 1 and a carrier protein;
preferably, the carrier protein is any one of keyhole limpet hemocyanin, bovine serum albumin, lactoferrin, horseradish peroxidase or ovalbumin.
5. The method of claim 4, wherein the method comprises: dissolving the opioid drug artificial hapten in an organic solvent, adding an activating agent, then adding a buffer solution dissolved with carrier protein, and reacting to obtain the opioid drug artificial antigen;
preferably, the organic solvent is N, N-dimethylformamide;
preferably, the activator is a mixture of N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide.
Preferably, the molar ratio of the opioid drug artificial hapten to the carrier protein is (25-100): 1, preferably 80: 1;
preferably, the molar ratio of the opioid drug artificial hapten to the N, N-dicyclohexylcarbodiimide and the N-hydroxysuccinimide is 1 (1-2) to (1-2), preferably 1:1.5: 1.5;
preferably, the buffer solution is a PBS solution;
preferably, the concentration of the PBS solution is 0.005-0.015 mol/L, preferably 0.01 mol/L;
preferably, the temperature of the reaction is 0-10 ℃, preferably 4 ℃; the reaction time is 10 to 20 hours, preferably 16 hours.
6. A polyclonal antibody, which is obtained by injecting the opioid drug artificial antigen according to claim 4 into an experimental animal and immunizing it for a plurality of times.
7. An enzyme-labeled antigen for opioid drugs, which is a conjugate of the opioid drug artificial hapten as claimed in claim 1 and horseradish peroxidase.
8. An immunological detection kit for opioid drugs, comprising the polyclonal antibody of claim 6 and the enzyme-labeled antigen for opioid drugs of claim 7.
9. Use of the opioid narcotic artificial antigen according to claim 4 or the polyclonal antibody according to claim 6 or the opioid narcotic enzyme-labeled antigen according to claim 7 or the opioid narcotic immunological detection kit according to claim 8 in the immunological detection of opioid narcotics;
preferably, the immunoassay for opioid narcotics is an immunoassay for opioid narcotics remaining in the hair.
10. An enzyme-linked immunoassay method for opioid narcotics in hair is characterized in that the assay method comprises the following steps: coating the polyclonal antibody of claim 6 on a microplate; adding the hair sample solution to be tested and the enzyme-labeled opium drug antigen of claim 7 into the micropores coated with the polyclonal antibody, and measuring the content of the opium drug in the sample to be tested by adopting an enzyme-linked immunosorbent assay method.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340748A (en) * | 1990-07-18 | 1994-08-23 | Abbott Laboratories | Analyte-substitute reagent for use in specific binding assay methods, devices and kits |
| CN107964030A (en) * | 2017-11-07 | 2018-04-27 | 宜昌人福药业有限责任公司 | A kind of synthetic method of morphine derivatives and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5340748A (en) * | 1990-07-18 | 1994-08-23 | Abbott Laboratories | Analyte-substitute reagent for use in specific binding assay methods, devices and kits |
| CN107964030A (en) * | 2017-11-07 | 2018-04-27 | 宜昌人福药业有限责任公司 | A kind of synthetic method of morphine derivatives and application |
Non-Patent Citations (2)
| Title |
|---|
| MASAO KOIDA ET AL.: ""THE MORPHINE 3-GLUCURONIDE DIRECTED ANTIBODY: ITS IMMUNOLOGICAL SPECIFICITY AND POSSIBLE USE FOR RADIOIMMUNOASSAY OF MORPHINE IN URINE"", 《JAPAN. J. PHARMACAL.》, vol. 1986, pages 707 - 714 * |
| PAUL P. DILLON ET AL.: ""Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor"", 《BIOSENSORS AND BIOELECTRONICS》, vol. 18, pages 217 - 227, XP055005163, DOI: 10.1016/S0956-5663(02)00182-3 * |
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