CN117865955A - Moxifloxacin derivative, homogeneous enzyme immunoassay reagent and preparation method thereof - Google Patents
Moxifloxacin derivative, homogeneous enzyme immunoassay reagent and preparation method thereof Download PDFInfo
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- FABPRXSRWADJSP-MEDUHNTESA-N moxifloxacin Chemical class COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 FABPRXSRWADJSP-MEDUHNTESA-N 0.000 title claims abstract description 223
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 109
- 238000002360 preparation method Methods 0.000 title claims abstract description 94
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- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 36
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- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 claims description 15
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- IMFACGCPASFAPR-UHFFFAOYSA-N tributylamine Chemical compound CCCCN(CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-N 0.000 claims description 6
- GVJXGCIPWAVXJP-UHFFFAOYSA-N 2,5-dioxo-1-oxoniopyrrolidine-3-sulfonate Chemical compound ON1C(=O)CC(S(O)(=O)=O)C1=O GVJXGCIPWAVXJP-UHFFFAOYSA-N 0.000 claims description 5
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Classifications
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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
- C07D471/04—Ortho-condensed systems
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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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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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
- G01N33/9446—Antibacterials
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Abstract
The invention discloses a moxifloxacin derivative, a homogeneous enzyme immunoassay reagent and a preparation method thereof, and relates to the technical field of biomedical detection. Use of the moxifloxacin derivative the moxifloxacin immunogen is prepared and obtained, and the moxifloxacin immunogen is used for immunizing experimental animals to prepare the anti-tumor drug the moxifloxacin specific antibody has no cross reaction with 108 common other medicines. The moxifloxacin enzyme-labeled conjugate is prepared by using the specific antibody against moxifloxacin the detection sensitivity of the moxifloxacin homogeneous enzyme immunoassay reagent reaches the pg/mL level, the method can accurately detect the concentration of trace moxifloxacin in the biological sample, and the detection accuracy, precision, sensitivity and specificity are obviously higher than those of the prior art.
Description
Technical Field
The invention relates to the technical field of biomedical detection, in particular to a moxifloxacin derivative, a homogeneous enzyme immunoassay reagent and a preparation method thereof.
Background
Therapeutic drug monitoring (Therapeutic Drug Monitoring, TDM) is based on the principle of clinical pharmacology, pharmacokinetics and clinical chemistry, using advanced drug analysis techniques, by measuring the concentration of a drug in blood or other body fluids, the related pharmacokinetic parameters are obtained, so that clinical safe and reasonable medication is guided, and a clinician is helped to formulate an individual medication scheme, so that the aims of avoiding or reducing toxic and side effects, improving the treatment effect and improving the cure rate are fulfilled. Moxifloxacin% Moxifloxacin is used as a carrier, MFX) is a fourth fluoroquinolone class of potent broad-spectrum antibacterial agents. WHO classified moxifloxacin as a core drug for the treatment of multi-drug resistant tuberculosis (MDR-TB) in 2016 and as a primary drug in the short-range treatment regimen for MDR-TB. The moxifloxacin can be rapidly absorbed without obvious first pass elimination effect, the absolute bioavailability can reach 90%, and the plasma half-life period is long and is about 11.4-15.6 hours. 400g of moxifloxacin taken orally 1 time a day makes it exceed the MIC90 of most pathogenic bacteria in most tissues, and the sterilization kinetics are characterized by concentration dependence, i.e. AUCo-24/MIC or Cmax/MIC, with AUCo-24/MIC being optimal. MFX has large inter-individual pharmacokinetic variability in guiding clinical medication for high risk groups such as multi-drug resistant pulmonary tuberculosis, severe and refractory tuberculosis, senile respiratory tract infection, children, pregnant women and the like or when used in combination with other drugs, and may cause low-dose drug exposure. The use of TDM assays can improve drug exposure and avoid unnecessary dose-related toxicity from empirically increasing dose. In addition, due to variability in AUC of moxifloxacin, variability in PK among critically ill patients, when MIC of pathogenic bacteria approaches drug sensitivity break point, and high tendency to develop drug resistance to quinolones, monitoring of drug concentration is also required to develop or adjust personalized dosing regimen to improve safety and effectiveness of moxifloxacin administration.
Examples of laboratory methods for detecting moxifloxacin include colorimetry, radioimmunoassay, high performance liquid chromatography, liquid tandem mass spectrometry, and enzyme-linked immunosorbent assay. Colorimetrically detect the total amount of moxifloxacin and the like rather than the level of a single compound; the radioimmunoassay has poor stability and has the problems of radiation emission, pollution and the like; the pretreatment of the high performance liquid chromatography is complex, and the target analyte is required to be completely separated on the chromatography, so that the analysis time is long; the liquid phase tandem mass spectrometry has high detection sensitivity, is rapid and quick, but has complex structure and high maintenance cost, needs to be operated by specially trained technicians, and is not easy to popularize in basic medical institutions; the ELISA method has high sensitivity and good specificity, but has long detection time and poor accuracy, and is difficult to be used for batch detection. Therefore, the moxifloxacin detection product which is wide in linear range, high in sensitivity, high in accuracy, high in precision, short in detection time, simple in sample processing, high in instrument automation degree and capable of continuously detecting multiple samples is lacking in the market at present.
Disclosure of Invention
The invention aims to provide a moxifloxacin derivative, a homogeneous enzyme immunoassay reagent and a preparation method thereof. The moxifloxacin homogeneous enzyme immunoassay reagent has the advantages of high sensitivity, good stability, strong specificity, accurate result and the like, can realize full-automatic rapid detection of a large number of samples, and overcomes the defects existing in the prior art.
The invention provides a moxifloxacin derivative, which has a structural formula shown in a formula (I):
the invention also provides a preparation method of the moxifloxacin derivative, which comprises the following synthetic route:
specifically, the preparation method of the moxifloxacin derivative comprises the following specific steps:
compound 1 (1.0 g,2.5 mmol) was dissolved in DCM (10 ml), then succinic anhydride (0.25 g,2.5 mmol) was added to make a reaction mixture solution, which was then stirred at room temperature for 12 hours; after the reaction, the reaction mixture solution is concentrated and then purified by flash chromatography to obtain the moxifloxacin derivative.
The resulting moxifloxacin derivative was chemically structurally identified by 1H NMR (Varian mercury plus MHz) spectroscopic scanning analysis (TMS as internal standard) with LC-MS (Agilent 1200A).
The invention also provides a moxifloxacin homogeneous enzyme immunoassay reagent, which comprises an R1 reagent and an R2 reagent;
the R1 reagent comprises an anti-moxifloxacin specific antibody and an R1 buffer solution;
the R2 reagent comprises a moxifloxacin enzyme-labeled conjugate and an R2 buffer solution;
the anti-moxifloxacin specific antibody is a polyclonal antibody generated after an experimental animal is immunized by moxifloxacin immunogen;
the experimental animal is a mammal; preferably, the mammal is one of a rabbit, sheep, goat, mouse, rat, guinea pig, donkey, horse or camel; more preferably, the mammal is a rabbit.
The moxifloxacin immunogen is a compound obtained by connecting a moxifloxacin derivative shown in a formula (I) with carrier protein, and the structural formula of the moxifloxacin immunogen is shown in a formula (II):
the carrier protein is protein with immunogenicity; preferably, the protein with immunogenicity is one of hemocyanin, serum protein, ovalbumin, gamma globulin, thyroglobulin or polylysine; more preferably, the protein having immunogenicity is horseshoe crab hemocyanin.
The R1 buffer solution is prepared from oxidized nicotinamide adenine dinucleotide, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative;
the moxifloxacin enzyme-labeled conjugate is formed by connecting a moxifloxacin derivative shown in a formula (I) with bacterial glucose-6-phosphate dehydrogenase, and the structural formula of the moxifloxacin enzyme-labeled conjugate is shown in a formula (III):
preferably, the bacterial glucose-6-phosphate dehydrogenase is one of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, leuconostoc citricola glucose-6-phosphate dehydrogenase, lactobacillus plantarum glucose-6-phosphate dehydrogenase, bifidobacterium lactis glucose-6-phosphate dehydrogenase, and Bacillus subtilis glucose-6-phosphate dehydrogenase; more preferably, the bacterial glucose-6-phosphate dehydrogenase is leuconostoc mesenteroides glucose-6-phosphate dehydrogenase.
The R2 buffer solution is prepared from glucose-6-phosphate, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative.
The invention also provides a preparation method of the moxifloxacin homogeneous enzyme immunoassay reagent, which comprises the following steps:
(A1) Preparation of R1 buffer: dissolving oxidized nicotinamide adenine dinucleotide, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative in purified water to prepare an R1 buffer solution;
(A2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody with the R1 buffer solution obtained in the step (A1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:50-5000;
(A3) Preparation of R2 buffer: dissolving glucose-6-phosphoric acid, tris, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative in purified water to prepare an R2 buffer solution;
(A4) Preparation of R2 reagent: and dissolving the moxifloxacin enzyme-labeled conjugate in an R2 buffer solution to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:50-5000.
Preferably, the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:750; the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:950.
Specifically, the preparation method of the moxifloxacin homogeneous enzyme immunoassay reagent comprises the following steps:
(A1) Preparation of R1 buffer: dissolving 5-15g of oxidized nicotinamide adenine dinucleotide, 10-80mg of tris, 0.5-2.5g of sodium chloride, 0.5-2.5g of magnesium chloride, 50-300mg of bovine serum albumin and 10-50mg of sodium azide preservative in 1-3L of purified water, and regulating the pH to 8.5 to prepare an R1 buffer solution;
(A2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody with the R1 buffer solution obtained in the step (A1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:50-5000;
(A3) Preparation of R2 buffer: dissolving 2-8g of glucose-6-phosphoric acid, 10-80mg of tris, 0.5-2.5g of sodium chloride, 0.5-2.5g of magnesium chloride, 50-300mg of bovine serum albumin and 10-50mg of sodium azide preservative in 1-3L of purified water, and regulating the pH to 8.0 to prepare an R2 buffer solution;
(A4) Preparation of R2 reagent: and (3) dissolving the moxifloxacin enzyme-labeled conjugate in the R2 buffer solution obtained in the step (A3) to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:50-5000.
Preferably, the preparation method of the moxifloxacin immunogen comprises the following steps:
(B1) Preparation of a carrier protein solution: dissolving carrier protein in phosphate buffer solution to obtain carrier protein solution;
(B2) Preparation of moxifloxacin derivative solution: mixing the moxifloxacin derivative, dimethylformamide, ethanol, potassium phosphate buffer solution, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolving to obtain a moxifloxacin derivative solution;
(B3) Synthesis of moxifloxacin immunogen: and (3) adding the moxifloxacin derivative solution obtained in the step (B2) into the carrier protein solution obtained in the step (B1), stirring for reaction, and dialyzing and purifying to obtain the moxifloxacin immunogen.
Specifically, the preparation method of the moxifloxacin immunogen comprises the following steps:
(B1) Preparation of limulus hemocyanin solution: dissolving Limulus hemocyanin in 0.2M potassium phosphate buffer solution (pH=8.5) to obtain Limulus hemocyanin solution with final concentration of 3-5 mg/mL;
(B2) Preparation of moxifloxacin derivative solution: mixing 100-300mg of the moxifloxacin derivative, 2-6mL of dimethylformamide, 2-6mL of ethanol, 3-10mL of potassium phosphate buffer solution (10 mM, pH=5.0), 100-300mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 20-80mg of N-hydroxysulfosuccinimide, and stirring and dissolving the mixture for 30-120min to obtain a moxifloxacin derivative solution;
(B3) Synthesis of moxifloxacin immunogen: adding the moxifloxacin derivative solution obtained in the step (B2) into the limulus blood blue protein solution obtained in the step (B1), stirring overnight at 0-8 ℃, and obtaining the moxifloxacin immunogen through dialysis and purification.
Preferably, the preparation method of the moxifloxacin-resistant specific antibody comprises the following steps:
(C1) Diluting the moxifloxacin immunogen with a phosphate buffer solution to obtain an artificial antigen solution, mixing the artificial antigen solution with an equivalent Freund complete adjuvant, and performing multi-point injection on the experimental animal;
(C2) After 3-6 weeks, mixing the same artificial antigen solution with equivalent Freund's incomplete adjuvant, performing multi-point injection on the experimental animals, and then injecting once every 3-6 weeks for 3-10 times in total;
(C3) And (3) taking blood from the experimental animal subjected to injection in the step (C2), and separating and purifying to obtain the moxifloxacin-resistant specific antibody.
Specifically, the preparation method of the moxifloxacin-resistant specific antibody comprises the following steps:
(C1) Diluting the moxifloxacin immunogen with 0.01M sodium phosphate buffer solution (pH=6.0) to a final concentration of 1.0-3.0mg/mL to obtain an artificial antigen solution, mixing the artificial antigen solution with equivalent Freund's complete adjuvant, and performing multi-point injection on experimental animal rabbits;
(C2) After 4 weeks, the experimental animal rabbits are subjected to multi-point injection by using the same artificial antigen solution and equivalent Freund incomplete adjuvant, and then the injection is carried out once every 4 weeks for 5-8 times in total;
(C3) And (3) taking blood from the experimental animal rabbits injected in the step (C2), and separating and purifying to obtain the anti-moxifloxacin specific antibody.
Preferably, the preparation method of the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(D1) Preparation of bacterial glucose-6-phosphate dehydrogenase solution: bacterial glucose-6-phosphate dehydrogenase and MgCl 2 Mixing with NaCl and dissolving in Tris buffer, adding reduced nicotinamide adenine dinucleotide, glucose-6-phosphate, carbitol and dimethyl sulfoxide, stirring and dissolving to obtain bacterial glucose-6-phosphate dehydrogenase solution;
(D2) Preparation of moxifloxacin derivative solution: dissolving the moxifloxacin derivative in dimethylformamide, cooling to below-4 ℃, adding tributyl amine and isobutyl chloroformate, and stirring and mixing uniformly to obtain a moxifloxacin derivative solution;
(D3) Synthesis of moxifloxacin enzyme-labeled conjugate: and (3) dropwise adding the moxifloxacin derivative solution obtained in the step (D2) into the bacterial glucose-6-phosphate dehydrogenase solution obtained in the step (D1), stirring for reaction, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
Specifically, the preparation method of the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(D1) Preparation of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution: mixing 5-20mg of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and 5-15mg of MgCl 2 Mixing with 50-150mg NaCl for dissolvingIn 5-20mL Tris buffer (1 m, ph=6.8); then adding 100-400mg of reduced nicotinamide adenine dinucleotide, 50-250mg of glucose-6-phosphoric acid, 0.3-1.5mL of carbitol and 1-3mL of dimethyl sulfoxide, stirring and dissolving to obtain leuconostoc mesenteroides glucose-6-phosphoric acid dehydrogenase solution;
(D2) Preparation of moxifloxacin derivative solution: dissolving 5-15mg of the moxifloxacin derivative in 300-900 mu L of dimethylformamide, cooling to-18 ℃, adding 1.5-4.5 mu L of tributylamine and 1-3 mu L of isobutyl chloroformate, and stirring and uniformly mixing to obtain a moxifloxacin derivative solution;
(D3) Preparation of moxifloxacin derivative solution: dropwise adding the moxifloxacin derivative solution obtained in the step (D2) into the leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution obtained in the step (D1), stirring and reacting at 2-8 ℃ overnight, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
The beneficial effects of the invention are as follows: the moxifloxacin immunogen prepared by using the moxifloxacin derivative and the anti-moxifloxacin specific antibody prepared by using the moxifloxacin immunogen immune experimental animal have no cross reaction with 108 common other medicines. The detection sensitivity of the moxifloxacin homogeneous enzyme immunoassay reagent prepared by using the moxifloxacin resistant specific antibody and the moxifloxacin enzyme-labeled conjugate reaches the pg/mL level, the concentration of trace moxifloxacin in a biological sample can be accurately detected, and the detection accuracy, precision, sensitivity and specificity are obviously higher than those of the prior art.
In addition, in order to prevent the glucose-6-phosphate dehydro-hapten enzyme-labeled conjugate from reacting with the enzyme substrate (glucose-6-phosphate) and coenzyme (nicotinamide adenine dinucleotide in an oxidized state) before using the homogeneous enzyme immunoassay reagent, the enzyme-labeled conjugate must be placed separately from the enzyme substrate and cannot be mixed. Therefore, the traditional homogeneous enzyme immunoassay reagent is to mix enzyme reaction substrate, coenzyme and hapten specific antibody together to prepare an R1 reagent, and glucose-6-phosphate dehydrogenation-hapten enzyme-labeled conjugate is added into the R2 reagent independently. That is, conventional homogeneous enzyme immunoassay reagents are mixed with the enzyme reaction substrate and the coenzyme prior to use. However, if a small amount of R2 reagent is mixed into R1 reagent by an operator carelessly before use, or if carrying pollution exists in the running process of the full-automatic biochemical analyzer, the enzyme and the enzyme reaction substrate react in advance, and under the condition that the enzyme reaction substrate and the coenzyme are sufficient, the reaction can continue, so that the enzyme reaction substrate is consumed in a large amount, and meanwhile, the oxidized coenzyme can be reduced into the reduced coenzyme in a large amount, so that the background signal of the detection reagent is continuously increased, the accuracy of the detection result is seriously influenced, and even the reagent is completely scrapped. The invention improves the formula of the traditional homogeneous enzyme immune reagent, adds the enzyme reaction substrate into the R2 reagent, only adds the antibody and the coenzyme into the R1 reagent, and does not add the enzyme reaction substrate any more, so that even if a small amount of R2 reagent is mixed into the R1 reagent before use, the reaction can be stopped immediately after the enzyme reaction substrate is consumed. Therefore, the invention can effectively avoid the background reaction signal rising caused by the cross contamination of the R1 reagent and the R2 reagent before the reagent is used, avoid the reagent waste and further improve the reagent stability and the detection accuracy.
Drawings
FIG. 1 is a standard graph of a moxifloxacin homogeneous enzyme immunoassay reagent in example 12 of the present invention;
FIG. 2 is a chart of 1H NMR spectroscopic scanning analysis of moxifloxacin derivatives of formula (I);
FIG. 3 is a LC-MS analysis spectrum of a moxifloxacin derivative of formula (I);
fig. 4 is a linear validation graph of the moxifloxacin homogeneous enzyme immunoassay reagent of example 16 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and detailed description, wherein it is to be understood that, on the premise of no conflict, the following embodiments or technical features may be arbitrarily combined to form new embodiments.
Example 1: preparation of moxifloxacin derivative
The synthetic route of the moxifloxacin derivative is as follows:
the preparation method of the moxifloxacin derivative comprises the following specific steps:
compound 1 (1.0 g,2.5 mmol) was dissolved in DCM (10 ml), then succinic anhydride (0.25 g,2.5 mmol) was added to make a reaction mixture solution, which was then stirred at room temperature for 12 hours; after the completion of the reaction, the reaction mixture solution was concentrated and then purified by flash chromatography to give moxifloxacin derivative (0.7 g).
The chemical structure of the moxifloxacin derivative obtained was identified by 1H NMR (Varian mercury plus MHz) spectroscopic scanning analysis (TMS as internal standard) and LC-MS (Agilent 1200A) (analytical spectra are shown in FIGS. 2 and 3).
Example 2: preparation of moxifloxacin immunogen
The preparation method of the moxifloxacin immunogen comprises the following steps:
(1) Preparation of a carrier protein solution: dissolving carrier protein in 0.2M potassium phosphate buffer (pH=8.5), wherein the final concentration of the carrier protein is 4mg/mL, so as to obtain carrier protein solution;
(2) Preparation of moxifloxacin derivative solution: 200mg of the above-mentioned moxifloxacin derivative, 4mL of dimethylformamide, 4mL of ethanol, 5mL of potassium phosphate buffer (10 mM, pH=5.0), 200mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, 50mg of N-hydroxysulfosuccinimide were mixed, stirring and dissolving the mixture for 90min to obtain moxifloxacin derivative solution;
(3) Synthesis of moxifloxacin immunogen: and (3) adding the moxifloxacin derivative solution obtained in the step (2) into the carrier protein solution obtained in the step (1), stirring overnight at 4 ℃, and obtaining the moxifloxacin immunogen through dialysis and purification.
Example 3: preparation of moxifloxacin immunogen
The preparation method of the moxifloxacin immunogen comprises the following steps:
(1) Preparation of a carrier protein solution: dissolving carrier protein in 0.2M potassium phosphate buffer (pH=8.5), wherein the final concentration of the carrier protein is 3mg/mL, so as to obtain carrier protein solution;
(2) Preparation of moxifloxacin derivative solution: mixing 100mg of the moxifloxacin derivative, 2mL of dimethylformamide, 2mL of ethanol, 3mL of potassium phosphate buffer solution (10 mM, pH=5.0), 100mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and 20mg of N-hydroxysulfosuccinimide, and stirring and dissolving the mixture for reaction for 30min to obtain a moxifloxacin derivative solution;
(3) Synthesis of moxifloxacin immunogen: and (3) adding the moxifloxacin derivative solution obtained in the step (2) into the carrier protein solution obtained in the step (1), stirring overnight at 0 ℃, and obtaining the moxifloxacin immunogen through dialysis and purification.
Example 4: preparation of moxifloxacin immunogen
The preparation method of the moxifloxacin immunogen comprises the following steps:
(1) Preparation of a carrier protein solution: dissolving carrier protein in 0.2M potassium phosphate buffer (pH=8.5), wherein the final concentration of the carrier protein is 5mg/mL, so as to obtain carrier protein solution;
(2) Preparation of moxifloxacin derivative solution: 300mg of the moxifloxacin derivative, 6mL of dimethylformamide, 6mL of ethanol, 10mL of potassium phosphate buffer (10 mM, pH=5.0) and 300mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide are mixed, and the mixture is stirred and dissolved for 120min to obtain a moxifloxacin derivative solution;
(3) Synthesis of moxifloxacin immunogen: and (3) adding the moxifloxacin derivative solution obtained in the step (2) into the carrier protein solution obtained in the step (1), stirring overnight at 8 ℃, and obtaining the moxifloxacin immunogen through dialysis and purification.
Example 5: preparation of anti-moxifloxacin specific antibody
The preparation method of the moxifloxacin-resistant specific antibody comprises the following steps of:
(1) Diluting the moxifloxacin immunogen with a 0.01M sodium phosphate buffer solution (pH=6.0) to a final concentration of 2.0mg/mL to obtain an artificial antigen solution, mixing the artificial antigen solution with an equivalent Freund complete adjuvant, and performing multi-point injection on experimental animal rabbits;
(2) After 4 weeks, the experimental animal rabbits are subjected to multi-point injection by using the same artificial antigen solution and equivalent Freund incomplete adjuvant, and then the injection is performed once every 4 weeks for 6 times in total;
(3) And (3) taking blood from the experimental animal rabbits injected in the step (2), and separating and purifying to obtain the anti-moxifloxacin specific antibody.
Example 6: preparation of anti-moxifloxacin specific antibody
The preparation method of the moxifloxacin-resistant specific antibody comprises the following steps of:
(1) Diluting the moxifloxacin immunogen with 0.01M sodium phosphate buffer solution (pH=6.0) to a final concentration of 1.0mg/mL to obtain an artificial antigen solution, mixing the artificial antigen solution with equivalent Freund's complete adjuvant, and performing multi-point injection on experimental animal rabbits;
(2) After 4 weeks, the experimental animal rabbits are subjected to multi-point injection by using the same artificial antigen solution and equivalent Freund incomplete adjuvant, and then the injection is carried out once every 4 weeks for 5 times in total;
(3) And (3) taking blood from the experimental animal rabbits injected in the step (2), and separating and purifying to obtain the anti-moxifloxacin specific antibody.
Example 7: preparation of anti-moxifloxacin specific antibody
The preparation method of the moxifloxacin-resistant specific antibody comprises the following steps of:
(1) Diluting the moxifloxacin immunogen with 0.01M sodium phosphate buffer solution (pH=6.0) to a final concentration of 3.0mg/mL to obtain an artificial antigen solution, mixing the artificial antigen solution with equivalent Freund's complete adjuvant, and performing multi-point injection on experimental animal rabbits;
(2) After 4 weeks, the experimental animal rabbits are subjected to multi-point injection by using the same artificial antigen solution and equivalent Freund incomplete adjuvant, and then the injection is performed once every 4 weeks for 8 times in total;
(3) And (3) taking blood from the experimental animal rabbits injected in the step (2), and separating and purifying to obtain the anti-moxifloxacin specific antibody.
Example 8: preparation of moxifloxacin enzyme-labeled conjugate
The preparation method of the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(1) Preparation of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution: 12mg of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and 10mg of MgCl are added 2 And 100mg NaCl in 12mL Tris buffer (1 m, ph=6.8); then 250mg of reduced nicotinamide adenine dinucleotide, 150mg of glucose-6-phosphoric acid, 0.8mL of carbitol and 2mL of dimethyl sulfoxide are added, stirred and dissolved to obtain a leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution;
(2) Preparation of moxifloxacin derivative solution: dissolving 10mg of the moxifloxacin derivative in 600 mu L of dimethylformamide, cooling to-18 ℃, adding 3.0 mu L of tributyl amine and 2 mu L of isobutyl chloroformate, and stirring and uniformly mixing to obtain a moxifloxacin derivative solution;
(3) Preparation of moxifloxacin derivative solution: and (3) dropwise adding the moxifloxacin derivative solution obtained in the step (2) into the leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution obtained in the step (1), stirring at 4 ℃ for reaction overnight, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
Example 9: preparation of moxifloxacin enzyme-labeled conjugate
The preparation method of the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(1) Preparation of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution: 5mg of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and 5mg of MgCl are added 2 And 50mg NaCl in 5mL Tris buffer (1 m, ph=6.8); then 100mg of reduced nicotinamide adenine dinucleotide, 50mg of glucose-6-phosphoric acid, 0.3mL of carbitol and 1mL of dimethyl sulfoxide are added, and stirring and dissolving are carried out, thus obtaining the leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution;
(2) Preparation of moxifloxacin derivative solution: dissolving 5mg of the moxifloxacin derivative in 300 mu L of dimethylformamide, cooling to-18 ℃, adding 1.5 mu L of tributylamine and 1 mu L of isobutyl chloroformate, and stirring and uniformly mixing to obtain a moxifloxacin derivative solution;
(3) Preparation of moxifloxacin derivative solution: dropwise adding the moxifloxacin derivative solution obtained in the step (2) into the leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution obtained in the step (1), stirring at 2 ℃ for reaction overnight, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
Example 10: preparation of moxifloxacin enzyme-labeled conjugate
The preparation method of the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(1) Preparation of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution: 20mg of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase and 15mg of MgCl are added 2 And 150mg NaCl in 20mL Tris buffer (1 m, ph=6.8); then 400mg of reduced nicotinamide adenine dinucleotide, 250mg of glucose-6-phosphoric acid, 1.5mL of carbitol and 3mL of dimethyl sulfoxide are added, stirred and dissolved to obtain a leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution;
(2) Preparation of moxifloxacin derivative solution: dissolving 15mg of the moxifloxacin derivative in 900 mu L of dimethylformamide, cooling to-18 ℃, adding 4.5 mu L of tributyl amine and 3 mu L of isobutyl chloroformate, and stirring and uniformly mixing to obtain a moxifloxacin derivative solution;
(3) Preparation of moxifloxacin derivative solution: and (3) dropwise adding the moxifloxacin derivative solution obtained in the step (2) into the leuconostoc mesenteroides glucose-6-phosphate dehydrogenase solution obtained in the step (1), stirring at 8 ℃ for reaction overnight, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
Example 11: preparation of moxifloxacin homogeneous enzyme immunoassay reagent
The preparation method of the moxifloxacin homogeneous enzyme immunoassay reagent comprises the following steps:
(1) Preparation of R1 buffer: 10g of oxidized nicotinamide adenine dinucleotide, 45mg of tris, 1.5g of sodium chloride, 1.5g of magnesium chloride, 150mg of bovine serum albumin and 30mg of sodium azide preservative are dissolved in 2L of purified water, and the pH is adjusted to 8.5 to prepare an R1 buffer solution;
(2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody prepared in the embodiment 5 with the R1 buffer solution obtained in the step (1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:750;
(3) Preparation of R2 buffer: dissolving 5g of glucose-6-phosphoric acid, 45mg of tris, 1.5g of sodium chloride, 1.5g of magnesium chloride, 150mg of bovine serum albumin and 30mg of sodium azide preservative in 2L of purified water, and adjusting the pH to 8.0 to prepare an R2 buffer;
(4) Preparation of R2 reagent: dissolving the moxifloxacin enzyme-labeled conjugate prepared in the embodiment 8 in the R2 buffer solution obtained in the step (3) to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:950.
Example 12: preparation of moxifloxacin homogeneous enzyme immunoassay reagent
The preparation method of the moxifloxacin homogeneous enzyme immunoassay reagent comprises the following steps:
(1) Preparation of R1 buffer: dissolving 5g of oxidized nicotinamide adenine dinucleotide, 10mg of tris, 0.5g of sodium chloride, 0.5g of magnesium chloride, 50mg of bovine serum albumin and 10mg of sodium azide preservative in 1L of purified water, and adjusting the pH to 8.5 to prepare an R1 buffer solution;
(2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody prepared in the embodiment 6 with the R1 buffer solution obtained in the step (1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:50;
(3) Preparation of R2 buffer: 2g of glucose-6-phosphoric acid, 10mg of tris, 0.5g of sodium chloride, 0.5g of magnesium chloride, 50mg of bovine serum albumin and 10mg of sodium azide preservative are dissolved in 1L of purified water, and the pH is adjusted to 8.0 to prepare an R2 buffer;
(4) Preparation of R2 reagent: dissolving the moxifloxacin enzyme-labeled conjugate prepared in the embodiment 9 in the R2 buffer solution obtained in the step (3) to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:50.
Example 13: preparation of moxifloxacin homogeneous enzyme immunoassay reagent
The preparation method of the moxifloxacin homogeneous enzyme immunoassay reagent comprises the following steps:
(1) Preparation of R1 buffer: dissolving 15g of oxidized nicotinamide adenine dinucleotide, 80mg of tris, 2.5g of sodium chloride, 2.5g of magnesium chloride, 300mg of bovine serum albumin and 50mg of sodium azide preservative in 3L of purified water, and adjusting the pH to 8.5 to prepare an R1 buffer solution;
(2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody prepared in the embodiment 7 with the R1 buffer solution obtained in the step (1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:5000;
(3) Preparation of R2 buffer: 8g of glucose-6-phosphoric acid, 80mg of tris, 2.5g of sodium chloride, 2.5g of magnesium chloride, 300mg of bovine serum albumin and 50mg of sodium azide preservative are dissolved in 3L of purified water, and the pH is adjusted to 8.0 to prepare an R2 buffer solution;
(4) Preparation of R2 reagent: dissolving the moxifloxacin enzyme-labeled conjugate prepared in the embodiment 10 in the R2 buffer solution obtained in the step (3) to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:5000.
Example 14: homogeneous enzyme immunoassay for moxifloxacin sample
(1) Establishing a moxifloxacin homogeneous enzyme immunoassay standard curve
The reaction parameters of the full-automatic biochemical analyzer are set according to the table 1, and the moxifloxacin homogeneous enzyme immunoassay reagent is the detection reagent prepared in the example 11. And adding the R1 reagent, then adding the standard substance, and finally adding the R2 reagent. After adding the R2 reagent, OD340 absorbance values at different time points are measured, reaction rates at different standard substance concentrations are calculated, and a reaction standard curve is drawn, as shown in FIG. 1.
Table 1: reaction parameters of full-automatic biochemical analyzer
(2) Moxifloxacin sample detection
The moxifloxacin sample is prepared by dissolving a moxifloxacin standard in artificial serum, and adjusting the concentration to be 1.50pg/mL, 3.00pg/mL and 6.00pg/mL respectively. The low, medium, high concentration moxifloxacin samples were repeatedly measured 10 times, the content of moxifloxacin in each sample was calculated according to the reaction standard curve shown in fig. 1, and the precision and recovery rate were calculated, recovery rate= (detection concentration average value/sample concentration) ×100%, and the results are shown in table 2.
Table 2: sample measurement and precision and recovery rate evaluation
Detection result: the moxifloxacin homogeneous enzyme immunoassay reagent has higher precision in determining moxifloxacin samples, and CV (constant velocity) is lower than 5%; the accuracy is higher, and the recovery rate reaches 95-105%.
In addition, the invention also uses the moxifloxacin homogeneous enzyme immunoassay reagent prepared in the examples 12 and 13 to carry out the homogeneous enzyme immunoassay of the moxifloxacin sample. The results show that: the detection reagent prepared by other embodiments of the invention has higher precision in moxifloxacin sample measurement, and CV is lower than 5%; the accuracy is high, and the recovery rate can reach 95-105%.
Example 15: cross-reaction experiments with other drugs
And selecting 108 common other medicines for cross reaction detection, dissolving a pure product of the medicine to be detected in artificial serum to prepare a medicine solution to be detected with the concentration of 100.00pg/mL, detecting the concentration of each medicine to be detected by using the moxifloxacin homogeneous enzyme immunoassay method described in the embodiment 14, and obtaining the concentration of the corresponding medicine according to a reaction standard curve shown in figure 1. 108 common other drug names and detection results are shown in Table 3.
Table 3: cross-reaction test results of other common drugs
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The experimental results show that: the actual detection values of the 108 common other drugs are 0.00pg/mL, so that the moxifloxacin homogeneous enzyme immunoassay reagent provided by the invention has strong specificity and does not have any cross reaction with the 108 common other drugs.
Example 16: linear verification of moxifloxacin homogeneous enzyme immunoassay reagent
Using a high value sample near the upper limit of the linear range and a low value sample near the lower limit of the linear range, mixing and preparing 7 samples with different concentrations (Xi) according to the EP6-A method, repeatedly measuring each concentration (Xi) 3 times to obtain a mean value (Yi) of a detection result, taking the diluted concentration (Xi) as an independent variable and taking the mean value (Yi) of the detection result as a dependent variable, and obtaining a linear regression equation (Table 4). The linear correlation coefficient r is calculated according to the formula to meet the standard requirement (figure 4).
Table 4: linear verification data of moxifloxacin homogeneous enzyme immunoassay reagent
Example 17: analysis sensitivity verification of moxifloxacin homogeneous enzyme immunoassay reagent
Taking purified water as a detection sample, and measuring the blank absorbance change value of the reagent; and meanwhile, a constant value sample with the moxifloxacin concentration of 20pg/mL is used as a specimen to measure the absorbance change value. The absolute value of the difference between the absorbance change value of the measured fixed value sample and the absorbance change value of the reagent blank was converted into the absolute value of the absorbance difference of the moxifloxacin sample of 20pg/mL (Table 5). Performance index requirements: the moxifloxacin sample with the concentration of 20pg/mL is measured, and the absorbance difference (delta A) is in the range of 0.01-0.40.
Table 5: analysis sensitivity verification data of moxifloxacin homogeneous enzyme immunoassay reagent
The above-described embodiments are only preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto, and equivalents and modifications made by those skilled in the art on the basis of the present invention are within the scope of the present invention, which is defined in the claims.
Claims (10)
1. The moxifloxacin derivative is characterized in that the structural formula of the moxifloxacin derivative is shown as the formula (I):
formula (I).
2. A process for the preparation of moxifloxacin derivatives as claimed in claim 1, characterized in that the synthetic route of the process is as follows:
。
3. the moxifloxacin homogeneous enzyme immunoassay reagent is characterized by comprising an R1 reagent and an R2 reagent;
the R1 reagent comprises an anti-moxifloxacin specific antibody and an R1 buffer solution;
the R2 reagent comprises a moxifloxacin enzyme-labeled conjugate and an R2 buffer solution;
the anti-moxifloxacin specific antibody is a polyclonal antibody generated after an experimental animal is immunized by moxifloxacin immunogen;
the experimental animal is a mammal;
the moxifloxacin immunogen is a compound obtained by connecting the moxifloxacin derivative of claim 1 with carrier protein, and the structural formula of the moxifloxacin immunogen is shown as formula (II):
formula (II);
the carrier protein is protein with immunogenicity;
the R1 buffer solution is prepared from oxidized nicotinamide adenine dinucleotide, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative;
the moxifloxacin enzyme-labeled conjugate is formed by connecting a moxifloxacin derivative according to claim 1 with bacterial glucose-6-phosphate dehydrogenase, and the structural formula of the moxifloxacin enzyme-labeled conjugate is shown as formula (III):
formula (III);
the R2 buffer solution is prepared from glucose-6-phosphate, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative.
4. A moxifloxacin homogeneous enzyme immunoassay reagent according to claim 3 wherein the mammal is one of a rabbit, sheep, goat, mouse, rat, guinea pig, donkey, horse or camel; the protein with immunogenicity is one of hemocyanin, serum protein, ovalbumin, gamma globulin, thyroglobulin or polylysine; the bacterial glucose-6-phosphate dehydrogenase is one of leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, leuconostoc citri glucose-6-phosphate dehydrogenase, lactobacillus plantarum glucose-6-phosphate dehydrogenase, bifidobacterium lactis glucose-6-phosphate dehydrogenase and bacillus subtilis glucose-6-phosphate dehydrogenase.
5. The moxifloxacin homogeneous enzyme immunoassay reagent of claim 4, wherein the mammal is a rabbit; the protein with immunogenicity is horseshoe crab hemocyanin; the bacterial glucose-6-phosphate dehydrogenase is Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase.
6. A method for preparing a moxifloxacin homogeneous enzyme immunoassay reagent according to any one of claims 3 to 5, wherein the method comprises the steps of:
(A1) Preparation of R1 buffer: dissolving oxidized nicotinamide adenine dinucleotide, tris (hydroxymethyl) aminomethane, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative in purified water to prepare an R1 buffer solution;
(A2) Preparation of R1 reagent: uniformly mixing the anti-moxifloxacin specific antibody described in claim 3 with the R1 buffer solution obtained in the step (A1) to obtain an R1 reagent, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:50-5000;
(A3) Preparation of R2 buffer: dissolving glucose-6-phosphoric acid, tris, sodium chloride, magnesium chloride, bovine serum albumin and sodium azide preservative in purified water to prepare an R2 buffer solution;
(A4) Preparation of R2 reagent: dissolving the moxifloxacin enzyme-labeled conjugate in the claim 3 in an R2 buffer solution to obtain an R2 reagent, wherein the volume ratio of the moxifloxacin enzyme-labeled conjugate in the R2 reagent to the R2 buffer solution is 1:50-5000.
7. The method for preparing the moxifloxacin homogeneous enzyme immunoassay reagent according to claim 6, wherein the method for preparing the moxifloxacin-resistant specific antibody comprises the following steps:
(B1) Diluting the moxifloxacin immunogen in claim 3 with phosphate buffer to obtain an artificial antigen solution, and then mixing the artificial antigen solution with equivalent Freund's complete adjuvant, and performing multi-point injection on the experimental animal in claim 3;
(B2) After 3-6 weeks, mixing the same artificial antigen solution with equivalent Freund's incomplete adjuvant, performing multi-point injection on the experimental animals, and then injecting once every 3-6 weeks for 3-10 times in total;
(B3) And (3) taking blood from the experimental animal subjected to injection in the step (B2), and separating and purifying to obtain the moxifloxacin-resistant specific antibody.
8. The method for preparing the moxifloxacin homogeneous enzyme immunoassay reagent according to claim 7, wherein the method for preparing the moxifloxacin immunogen comprises the following steps:
(C1) Preparation of a carrier protein solution: dissolving carrier protein in phosphate buffer solution to obtain carrier protein solution;
(C2) Preparation of moxifloxacin derivative solution: mixing the moxifloxacin derivative of claim 1 with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysulfosuccinimide, and stirring for dissolution to obtain a moxifloxacin derivative solution;
(C3) Synthesis of moxifloxacin immunogen: and (3) adding the moxifloxacin derivative solution obtained in the step (C2) into the carrier protein solution obtained in the step (C1), stirring for reaction, and dialyzing and purifying to obtain the moxifloxacin immunogen.
9. The method for preparing the moxifloxacin homogeneous enzyme immunoassay reagent according to claim 6, wherein the method for preparing the moxifloxacin enzyme-labeled conjugate comprises the following steps:
(D1) Preparation of bacterial glucose-6-phosphate dehydrogenase solution: bacterial glucose-6-phosphate dehydrogenase and MgCl 2 Mixing with NaCl and dissolving in Tris buffer, adding reduced nicotinamide adenine dinucleotide, glucose-6-phosphate, carbitol and dimethyl sulfoxide, stirring and dissolving to obtain bacterial glucose-6-phosphate dehydrogenase solution;
(D2) Preparation of moxifloxacin derivative solution: dissolving the moxifloxacin derivative according to claim 1 in dimethylformamide, cooling to below-4 ℃, adding tributyl amine and isobutyl chloroformate, and stirring and mixing uniformly to obtain a moxifloxacin derivative solution;
(D3) Synthesis of moxifloxacin enzyme-labeled conjugate: and (3) dropwise adding the moxifloxacin derivative solution obtained in the step (D2) into the bacterial glucose-6-phosphate dehydrogenase solution obtained in the step (D1), stirring for reaction, and purifying by a gel chromatographic column to obtain the moxifloxacin enzyme-labeled conjugate.
10. The method for preparing moxifloxacin homogeneous enzyme immunoassay reagent according to claim 6, wherein the volume ratio of the anti-moxifloxacin specific antibody to the R1 buffer solution in the R1 reagent is 1:750; the volume ratio of the moxifloxacin enzyme-labeled conjugate to the R2 buffer solution in the R2 reagent is 1:950.
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