Vancomycin chemiluminescence immunoassay reagent and preparation and detection method thereof

Abstract

The application discloses a vancomycin chemiluminescence immunoassay reagent and a preparation and detection method thereof. The immunoreagent developed by using a high-titer anti-vancomycin specific antibody can accurately determine the content of vancomycin in serum and plasma samples. Compared with traditional methods, the vancomycin chemiluminescence immunoassay reagent provided by the application has high sensitivity and strong specificity, and is combined with a chemiluminescence immunoassay analyzer, and has the advantages of simple operation, high throughput, accurate detection results and the like, and has important significance for guiding clinical rational drug use.

Description

Technical Field

[0001] This invention belongs to the field of biomedical testing, specifically relating to a vancomycin chemiluminescent immunoassay reagent and its preparation and detection method. Background Technology

[0002] Therapeutic Drug Monitoring (TDM) is a clinical drug analysis technique guided by principles of clinical pharmacology, pharmacokinetics, and clinical chemistry. It utilizes advanced drug analysis techniques to measure drug concentrations in blood or other body fluids, obtaining relevant pharmacokinetic parameters to guide safe and rational drug use in clinical practice. This helps clinicians develop individualized dosing regimens to avoid or reduce side effects, improve treatment efficacy, and increase cure rates. Vancomycin is a tricyclic glycopeptide antibiotic that disrupts the integrity of bacterial cell walls by interfering with the cross-linking synthesis of peptidoglycan, a key component of bacterial cell wall structure. Its molecular formula is C66H75C12N9O24, and its molecular weight is 1485.74. Vancomycin is primarily used to treat infections caused by Gram-positive bacteria and certain specific Gram-negative anaerobic bacteria. It is a first-line drug for treating methicillin-resistant Staphylococcus aureus (MRSA) infections. Adverse reactions to vancomycin mainly include ototoxicity, nephrotoxicity, and red man syndrome, followed by gastrointestinal reactions, thrombocytopenia, or leukopenia. The incidence and severity of these adverse reactions are related to blood drug concentration. The metabolism of vancomycin in the body is complex, and individual differences can significantly affect blood drug concentration. Infants, the elderly, patients with impaired liver or kidney function, or those with higher body weight are more likely to have increased blood drug concentrations; while young children and patients with severe infections will have relatively lower concentrations. Furthermore, combined clinical medications can also affect vancomycin blood concentrations. For example, imipenem-cilastatin, fluoxetine, and fosfomycin can increase clearance and decrease blood drug concentrations. Therefore, scientific monitoring of vancomycin blood concentrations is crucial, as it helps in developing and adjusting individualized dosing regimens, thereby improving efficacy and further ensuring medication safety.

[0003] Currently, the main laboratory methods for detecting vancomycin include microbial assays, radioimmunoassay (RIA), fluorescence polarization immunoassay (FPIA), chemiluminescent microparticle immunoassay (CMIA), high-performance liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). These methods all have their unavoidable drawbacks. For example, HPLC is often limited in its widespread application in clinical drug concentration monitoring due to its cumbersome operation, low efficiency, long measurement cycle, and high analytical cost.

[0004] This invention employs a competitive immunoassay principle, using magnetic microparticles as the solid phase of the immunoassay reaction. It utilizes chemiluminescence immunoassay in conjunction with a fully automated chemiluminescence immunoassay analyzer to determine the vancomycin content in human samples. The sample, vancomycin-alkaline phosphatase conjugate, and paramagnetic particles coated with anti-vancomycin specific antibodies are added to the reaction vessel. Vancomycin in the sample competes with the vancomycin-alkaline phosphatase conjugate for a limited number of binding sites on the anti-vancomycin specific antibody. After incubation at 37°C in the reaction vessel, vancomycin and the vancomycin-alkaline phosphatase conjugate bound to the specific antibody on the solid-phase magnetic beads are held in the magnetic field, while unbound vancomycin and the vancomycin-alkaline phosphatase conjugate are washed away. Then, substrate solution for the fully automated immunoassay system is injected, and the intensity of the chemiluminescent photons is detected. The intensity of the light is inversely proportional to the concentration of vancomycin in the sample. The amount of analyte in the sample is determined by a stored multi-point calibration curve. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the purpose of this invention is to provide a detection reagent capable of high-throughput and rapid detection of vancomycin on a fully automated chemiluminescence immunoassay analyzer, in order to meet the growing domestic demand for detection reagents.

[0006] The technical solution adopted by the present invention to solve its technical problem is as follows: The first aspect of the present invention provides a vancomycin derivative, the structural formula of which is shown in the following formula (Ⅰ):

[0007]

[0008] Wherein, R1 is a linking group, and the linking group is -CO-(CH2). n -COOH, -(CH2) n -COOH, -O-(CH2) n -COOH, -S-(CH2) n -COOH, -NH-(CH2) n -COOH, -CS-(CH2) n -COOH, -(CH2) n -NH-COOH or One of them;

[0009] Where n is any integer between 1 and 10, including 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and more preferably, n is 3.

[0010] In the vancomycin derivatives with the above structural formula as shown in formula (Ⅰ), preferably, the linking group R1 is -CO-(CH2). n-COOH; where n is any integer between 1 and 10, including 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, and more preferably, n is 3.

[0011] Furthermore, more preferably, in the vancomycin derivative with the above-mentioned structural formula as shown in formula (I), the linking group R1 is -CO-(CH2)3-COOH.

[0012] In another preferred embodiment of the present invention, a vancomycin derivative is provided, the structural formula of which is shown in formula (II):

[0013]

[0014] A second aspect of the present invention also provides a method for preparing a vancomycin derivative with the structural formula shown in formula (I), comprising reacting compound 1 with compound R1-X, which is capable of introducing the above-mentioned linking group R1, to prepare:

[0015]

[0016] In the above method, preferably, compound 1, compound R1-X, and triethylamine are dissolved in dimethylformamide, the solution is stirred at room temperature, and the resulting reaction product is purified. Preferably, high performance liquid chromatography is used for concentration and purification to obtain the vancomycin derivative shown in formula (I). The compound R1-X is a corresponding organic acid or acid anhydride that can introduce the above-mentioned linking group R1.

[0017] More preferably, compound 1, compound R1-X and triethylamine are added sequentially to dimethylformamide for dissolution, wherein the molar ratio of compound 1 to compound R1-X is 1:1 to 1.5, preferably 1:1.1; the molar ratio of compound 1 to triethylamine is 1:2 to 2.5. The solution is stirred at room temperature for an appropriate time (e.g., 10 to 12 h), and the resulting reaction product is concentrated and purified by high performance liquid chromatography to obtain the vancomycin derivative shown in formula (I).

[0018] Furthermore, the present invention provides a method for preparing the vancomycin derivative shown in formula (II) comprising the following steps:

[0019]

[0020] Compound 1, glutaric anhydride and triethylamine were added to dimethylformamide and dissolved. The solution was stirred at room temperature. After the reaction was completed, the reaction product was purified to obtain the vancomycin derivative shown in formula (II).

[0021] Preferably, after the reaction is complete, the reaction product obtained is concentrated and purified by high performance liquid chromatography;

[0022] More preferably, the molar ratio of compound 1 to glutaric anhydride is 1:1 to 1.5, more preferably 1:1.1; and the molar ratio of compound 1 to triethylamine is 1:2 to 2.5.

[0023] In a more specific embodiment of the present invention, a method for preparing the vancomycin derivative of formula (II) is provided, comprising the following steps:

[0024] 5g of compound 1, 433mg of glutaric anhydride and 697mg of triethylamine were added sequentially to 5mL of dimethylformamide and dissolved. The solution was stirred at room temperature for 12 hours. The reaction product was concentrated and purified by high performance liquid chromatography to obtain the vancomycin derivative shown in formula (II).

[0025] In a third aspect, the present invention provides a vancomycin artificial antigen, which is formed by linking a vancomycin derivative shown in the aforementioned structural formula (I) with a carrier, the structural formula of which is shown in formula (III):

[0026]

[0027] Wherein, R2 is a linking group, and the linking group is -CO-(CH2). n -CO-, -(CH2) n -CO-, -O-(CH2) n -CO-, -S-(CH2) n -CO-, -NH-(CH2) n -CO-, -CS-(CH2) n -CO-, -(CH2) n -NH-CO- or One of them;

[0028] Where n is any integer between 1 and 10, preferably n is 3;

[0029] In the above structural formula (Ⅲ), more preferably, the linking group is -CO-(CH2). n -CO-, where n is any integer between 1 and 10, preferably n is 3;

[0030] More preferably, in the above structural formula (Ⅲ), the linking group is -CO-(CH2)3-CO-;

[0031] The carrier is one of serum albumin, immunoglobulin, fibrinogen, ovalbumin, thyroglobulin, hemocyanin, and polylysine, preferably a serum protein, and more preferably bovine serum albumin.

[0032] In a fourth aspect, the present invention provides an anti-vancomycin specific antibody, which is isolated and purified from experimental animals after immunization with a vancomycin artificial antigen with the structural formula shown in Formula (III).

[0033] Furthermore, the anti-vancomycin specific antibody is a polyclonal antibody obtained by boosting the immunization of experimental animals with the vancomycin artificial antigen with the structural formula shown in Formula (III), or a monoclonal antibody obtained by somatic cell hybridization after immunizing experimental animals with the vancomycin artificial antigen with the structural formula shown in Formula (III); the host animal is one of rabbit, goat, sheep, rat, mouse, guinea pig or horse.

[0034] In a fifth aspect, the present invention provides a vancomycin chemiluminescent immunoassay reagent containing an anti-vancomycin specific antibody and an indicator reagent; the indicator reagent is selected from chemiluminescent reagents, enzyme reagents, radioisotope reagents, and fluorescent reagents; preferably, the indicator reagent is a chemiluminescent reagent composed of a vancomycin-alkaline phosphatase conjugate and a luminescent substrate, wherein the luminescent substrate is 2-chloro-5-(4-methoxyspiro-1,2-dioxane-3,2′-(5-chlorotricyclo3.3.1.13.7decane)-4-yl-1-phenylphosphate disodium salt.

[0035] In a sixth aspect, the present invention provides a method for preparing a vancomycin chemiluminescent immunoassay reagent, comprising the following steps:

[0036] Preparation of S1.1 Chemiluminescent reagent 1: Magnetic microparticles coated with anti-vancomycin specific antibody were added to the magnetic bead base solution until the final concentration of the magnetic microparticles coated with anti-vancomycin specific antibody was 0.1 mg / mL. The mixture was vortexed for 1 minute. The pH of the solution was adjusted to 7.10-7.30 with 6N HCl or 3M NaOH. The volume was brought up to 1L with the magnetic bead base solution. After the volume was brought up, the mixture was stirred for another 10 minutes.

[0037] Preparation of S1.2 Chemiluminescent reagent 2: Add vancomycin-alkaline phosphatase conjugate to alkaline phosphatase base solution until the final concentration of vancomycin-alkaline phosphatase conjugate is 1 KU / L. Stir with a magnetic stirrer for 25-35 minutes. Adjust the pH of the solution to 7.50-7.70 with 6N HCl or 3M NaOH. Make up the volume to 1L with alkaline phosphatase base solution. After making up the volume, continue stirring for ten minutes.

[0038] Preparation of S1.3 Chemiluminescent reagent 3: Add 90% purified water (v / v), 8.47g of 145mmol / L sodium chloride, and 0.5mL of 0.05% preservative Precursor Pryknin 300. Stir and mix for 25-35 minutes. Adjust the pH of the solution to 7.10-7.30 with 6N HCl or 3M NaOH. Make up the volume to 1L. After making up the volume, continue stirring for ten minutes.

[0039] The preparation of the anti-vancomycin specific antibody in the above method includes the following steps:

[0040] A. Place the vancomycin derivative shown in structural formula (I) in a container, and add dimethylamide, ethanol, potassium phosphate buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxythiosuccinimide in sequence, and stir to dissolve;

[0041] B. Dissolve bovine serum albumin in phosphate buffer;

[0042] C. Add the activated vancomycin derivative from step A to the bovine serum albumin solution obtained in step B, stir at 2–8°C for 8–24 hours, and dialyze the resulting solution using a dialysis bag to purify it; thus, prepare the vancomycin artificial antigen.

[0043] D. Dilute the vancomycin artificial antigen synthesized in step C with phosphate buffer, mix the diluted vancomycin artificial antigen solution with Freund's complete adjuvant, and inject it into experimental animals. 14-28 days later, mix the same vancomycin artificial antigen solution with Freund's incomplete adjuvant and inject it into the experimental animals once more. Thereafter, inject once every 14-28 days. After 3-6 months from the first injection, collect blood from the experimental animals, separate and purify the antibodies.

[0044] Preferably, in the above method, the preparation of the anti-vancomycin specific antibody includes the following steps:

[0045] A. Place 10 mg of the vancomycin derivative shown in structural formula (I) in a container, and add dimethylamide, ethanol, potassium phosphate buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxythiosuccinimide in sequence, and stir to dissolve;

[0046] Preferably, the molar ratio of the vancomycin derivative shown in structural formula (I) to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:3-5, and the molar ratio of the vancomycin derivative shown in structural formula (I) to N-hydroxythiosuccinimide is 1:35-45; the volume ratio of dimethylamide:ethanol:potassium phosphate buffer is 1:1.5-2:2-3, the concentration of the potassium phosphate buffer is 10 mmol / L, and the pH value is 5.5-6.5;

[0047] B. Dissolve bovine serum albumin in phosphate buffer solution at pH 8.5;

[0048] Preferably, the concentration of the phosphate buffer is 0.2 mol / L;

[0049] C. Add the activated vancomycin derivative from step A to the bovine serum albumin solution obtained in step B, stir at 2-8°C for 12-16 hours, and dialyze the resulting solution using a dialysis bag with a pore size of 8KD to obtain vancomycin artificial antigen.

[0050] D. The vancomycin artificial antigen synthesized in step C was diluted to 1.0 mg / mL with 10 mmol / L, pH 7.4 phosphate buffer. The vancomycin artificial antigen solution was then mixed with Freund's complete adjuvant and injected into rabbits. After 21 days, the same vancomycin artificial antigen solution was mixed with Freund's incomplete adjuvant and injected into rabbits once more. Subsequent injections were given every 21 days. After 4 months from the initial injection, blood was collected from the rabbits, and antibodies were separated and purified.

[0051] The preparation method of the magnetic microparticles coated with anti-vancomycin specific antibody in the above method is as follows: 10 mL of 5 mg / mL magnetic beads, 90 mL of activation solution, 20 μg / mg of anti-vancomycin specific antibody, 500 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 500 mg / mL of N-hydroxythiosuccinimide sodium salt are placed in a rotary incubator and mixed by rotation. The magnetic beads are coated for 120 minutes. The coated magnetic beads are washed 3 times with washing solution. 100 mL of magnetic bead blocking solution is added and mixed by rotation. The magnetic beads are blocked for 60 minutes. The blocked magnetic beads are washed 3 times with washing solution. 100 mL of magnetic bead base solution is added to the washed magnetic beads to preserve them.

[0052] The magnetic bead blocking solution is prepared as follows: 90% purified water (volume fraction), 20.0g of 2% bovine serum albumin, 0.5mL of 0.05% Tween-20, 0.5mL of 0.05% preservative Precambriconazole 300, and 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.10–7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0053] The cleaning solution is prepared as follows: 90% purified water (volume fraction), 8.5g of 145mmol / L sodium chloride, 0.95g of 6.66mmol / L disodium hydrogen phosphate, 0.45g of 3.33mmol / L potassium dihydrogen phosphate, 0.5mL of 0.05% preservative Precinctine 300, and 0.5mL of 0.05% Tween-20 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.10-7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0054] The preparation method of the magnetic bead base solution is as follows: 90% purified water, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, 2.5g of 0.25% bovine serum albumin, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 6.90-7.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0055] The vancomycin-alkaline phosphatase conjugate is prepared as follows: 5.6 μL of 1 KU / mL alkaline phosphatase is added to 0.5 mL of alkaline phosphatase basal solution to prepare an alkaline phosphatase solution. 2 mg of 2 mg / mL vancomycin derivative, 0.5 mL of activation solution, 2 mg / mL of 2 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2 mg / mL of 2 mg / mL N-hydroxythiosuccinimide sodium salt are placed in a magnetic stirrer and stirred for 5 minutes to activate. The activated vancomycin derivative is added to the above alkaline phosphatase solution and coupled at 2-8℃ for 120 minutes. The coupled vancomycin-alkaline phosphatase conjugate is dialyzed 4 times with dialysis buffer, each dialyzing for 8 hours.

[0056] The alkaline phosphatase base solution is prepared as follows: 90% purified water (v / v), 1.25g of 0.125% bovine serum albumin, 0.20g of 1mmol / L magnesium chloride, 13.63mg of 0.1mmol / L zinc chloride, 8.5g of 145.5mmol / L sodium chloride, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.50–7.70 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0057] The preparation method of the activation solution is as follows: 90% purified water (volume fraction), 19.52g of 100mmol / L morpholine ethanesulfonic acid, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 4.90-5.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0058] The preparation method of the dialysate is as follows: 90% purified water, 8.47g of 145mmol / L sodium chloride, 1.15g of 8.1mmol / L disodium hydrogen phosphate, 0.2g of 1.47mmol / L potassium dihydrogen phosphate, and 0.5mL of 0.05% preservative are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.30-7.50 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0059] The seventh aspect of the present invention provides a detection method for a vancomycin chemiluminescent immunoassay reagent, wherein the vancomycin chemiluminescent immunoassay reagent is assembled into a kit product, the kit product being used to determine the vancomycin content in a biological sample, the biological sample being a liquid physiological sample of a human or animal, the liquid physiological sample being serum, plasma, whole blood, urine, saliva, lymph, tissue fluid, cerebrospinal fluid, amniotic fluid, or tears.

[0060] This invention provides a novel vancomycin derivative and its preparation method. The anti-vancomycin specific antibody developed using this novel vancomycin derivative can be used to prepare a vancomycin immunoassay reagent with high sensitivity, strong specificity, and good detection effect. This invention also provides a method for preparing the vancomycin immunoassay reagent and a corresponding detection method. The vancomycin chemiluminescent immunoassay reagent provided by this invention is convenient to use, rapid in detection, accurate in results, highly sensitive, and highly specific, and can quantitatively detect the vancomycin content in human serum, plasma, and other samples. It overcomes the shortcomings of existing vancomycin detection methods, such as complex operation and low degree of automation, and can effectively guide personalized and rational drug use in clinical practice. Attached Figure Description

[0061] Figure 1 This is the standard curve of vancomycin chemiluminescent immunoassay.

[0062] Figure 2 This is a linear analysis graph of the vancomycin chemiluminescent immunoassay. Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These drawings are simplified schematic diagrams, illustrating only the basic structure of the invention and therefore only showing the components relevant to the invention. Unless otherwise specified, the reagents, instruments, equipment, and consumables used in the following embodiments can be purchased from legitimate suppliers.

[0064] Example 1: Preparation of vancomycin derivatives

[0065] The structural formula of vancomycin derivatives is shown in formula (II):

[0066]

[0067] The specific preparation steps for the above-mentioned vancomycin derivatives are as follows:

[0068]

[0069] 5g of compound 1, 433mg of glutaric anhydride and 697mg of triethylamine were dissolved in 5mL of dimethylformamide. The solution was stirred at room temperature for 12 hours. The reaction product was concentrated and purified by high performance liquid chromatography to obtain 1g of white solid vancomycin derivative, with a yield of 18.5%.

[0070] Vancomycin derivatives of formula (I) were prepared using a similar method when R1 was another linking group as described in this invention. Example 2: Preparation of vancomycin immunoassay reagent

[0071] The specific preparation steps for the vancomycin chemiluminescent immunoassay reagent are as follows:

[0072] Preparation of S1.1 Chemiluminescent reagent 1: Magnetic microparticles coated with anti-vancomycin specific antibody were added to the magnetic bead base solution until the final concentration of the magnetic microparticles coated with anti-vancomycin specific antibody was 0.1 mg / mL. The mixture was vortexed for 1 minute. The pH of the solution was adjusted to 7.10-7.30 with 6N HCl or 3M NaOH. The volume was brought up to 1L with the magnetic bead base solution. After the volume was brought up, the mixture was stirred for another 10 minutes.

[0073] Preparation of S1.2 Chemiluminescent reagent 2: Add vancomycin-alkaline phosphatase conjugate to alkaline phosphatase base solution until the final concentration of vancomycin-alkaline phosphatase conjugate is 1 KU / L. Stir with a magnetic stirrer for 25-35 minutes. Adjust the pH of the solution to 7.50-7.70 with 6N HCl or 3M NaOH. Make up the volume to 1L with alkaline phosphatase base solution. After making up the volume, continue stirring for ten minutes.

[0074] Preparation of S1.3 Chemiluminescent reagent 3: Add 90% purified water (v / v), 8.47g of 145mmol / L sodium chloride, and 0.5mL of 0.05% preservative Precursor Pryknin 300. Stir and mix for 25-35 minutes. Adjust the pH of the solution to 7.10-7.30 with 6N HCl or 3M NaOH. Make up the volume to 1L. After making up the volume, continue stirring for ten minutes.

[0075] The preparation of the anti-vancomycin specific antibody includes the following specific steps:

[0076] A. Place 10 mg of the vancomycin derivative prepared in Example 1 into a container, and add 2.5 mL of dimethylamide, 4.5 mL of ethanol, 7 mL of potassium phosphate buffer, 400 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 50 mg of N-hydroxythiosuccinimide in sequence; the potassium phosphate buffer has a concentration of 10 mmol / L and a pH of 5.5;

[0077] B. Dissolve 100 mg of bovine serum albumin in 100 ml of 0.2 mol / L phosphate buffer at pH 8.5;

[0078] C. Add the activated vancomycin derivative from step A to the bovine serum albumin solution obtained in step B, stir at 2-8°C for 12-16 hours, and dialyze the resulting solution using a dialysis bag with a pore size of 8KD to obtain vancomycin artificial antigen.

[0079] D. The vancomycin artificial antigen synthesized in step C was diluted to 1.0 mg / mL with 10 mmol / L, pH 7.4 phosphate buffer. The vancomycin artificial antigen solution was then mixed with Freund's complete adjuvant and injected into rabbits. After 21 days, the same vancomycin artificial antigen solution was mixed with Freund's incomplete adjuvant and injected into rabbits once more. Subsequent injections were given every 21 days. After 4 months from the initial injection, blood was collected from the rabbits, and antibodies were separated and purified.

[0080] The preparation method of the magnetic microparticles coated with anti-vancomycin specific antibody is as follows: 10 mL of 5 mg / mL magnetic beads, 90 mL of activation solution, 20 μg / mg of anti-vancomycin specific antibody, 500 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 500 mg / mL of N-hydroxythiosuccinimide sodium salt are placed in a rotary incubator and mixed by rotation. The magnetic beads are coated for 120 minutes. The coated magnetic beads are washed three times with washing solution. 100 mL of magnetic bead blocking solution is added and mixed by rotation. The magnetic beads are blocked for 60 minutes. The blocked magnetic beads are washed three times with washing solution. 100 mL of magnetic bead base solution is added to the washed magnetic beads to preserve them.

[0081] The magnetic bead blocking solution is prepared as follows: 90% purified water (volume fraction), 20.0g of 2% bovine serum albumin, 0.5mL of 0.05% Tween-20, 0.5mL of 0.05% preservative Precambriconazole 300, and 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.10–7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0082] The cleaning solution is prepared as follows: 90% purified water (volume fraction), 8.5g of 145mmol / L sodium chloride, 0.95g of 6.66mmol / L disodium hydrogen phosphate, 0.45g of 3.33mmol / L potassium dihydrogen phosphate, 0.5mL of 0.05% preservative Precambriconazole 300, and 0.5mL of 0.05% Tween-20 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.10-7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0083] The preparation method of the magnetic bead base solution is as follows: 90% purified water, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, 2.5g of 0.25% bovine serum albumin, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 6.90-7.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0084] The vancomycin-alkaline phosphatase conjugate is prepared as follows: 5.6 μL of 1 KU / mL alkaline phosphatase is added to 0.5 mL of alkaline phosphatase basal solution to prepare an alkaline phosphatase solution. 2 mg of 2 mg / mL vancomycin derivative, 0.5 mL of activation solution, 2 mg / mL of 2 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2 mg / mL of 2 mg / mL N-hydroxythiosuccinimide sodium salt are placed in a magnetic stirrer and stirred for 5 minutes to activate. The activated vancomycin derivative is added to the above alkaline phosphatase solution and coupled at 2-8℃ for 120 minutes. The coupled vancomycin-alkaline phosphatase conjugate is dialyzed 4 times with dialysis buffer, each dialyzing for 8 hours.

[0085] The alkaline phosphatase base solution is prepared as follows: 90% purified water (v / v), 1.25g of 0.125% bovine serum albumin, 0.20g of 1mmol / L magnesium chloride, 13.63mg of 0.1mmol / L zinc chloride, 8.5g of 145.5mmol / L sodium chloride, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.50–7.70 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes.

[0086] The preparation method of the activation solution is as follows: 90% purified water (volume fraction), 19.52g of 100mmol / L morpholine ethanesulfonic acid, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 4.90-5.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0087] The preparation method of the dialysate is as follows: 90% purified water, 8.47g of 145mmol / L sodium chloride, 1.15g of 8.1mmol / L disodium hydrogen phosphate, 0.2g of 1.47mmol / L potassium dihydrogen phosphate, and 0.5mL of 0.05% preservative are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.30-7.50 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

[0088] Example 3: Vancomycin Detection in Samples Using Vancomycin Immunoassay Reagent

[0089] (1) Set the reaction parameters of the fully automated chemiluminescence immunoassay analyzer (Table 1).

[0090] Table 1. Reaction parameters for vancomycin detection using a fully automated chemiluminescence immunoassay analyzer.

[0091]

[0092] (2) Reagent performance evaluation experiment

[0093] The performance of the vancomycin chemiluminescent immunoassay reagent prepared in Example 2 was validated. The main performance indicators tested included appearance, fill volume, limit of detection, linearity, repeatability, batch-to-batch difference, accuracy, calibrator accuracy and homogeneity, and quality control accuracy and homogeneity. Experimental parameters were set according to the methods described above. The reagent performance validation results are as follows:

[0094] Appearance

[0095]

[0096]

[0097]

[0098] Fill volume

[0099] Fill volume Detection standard determination Vancomycin R1: 5.2mL, R2: 5.1mL, R3: 10.2mL R1≥5.0mL, R2≥5.0mL, R3≥10.0mL conform to

[0100] Fill volume

[0101]

[0102] Fill volume

[0103]

[0104] Unit: Limit of Detection (μg / mL)

[0105]

[0106] Linearity: 5.00–80.00 μg / mL

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114] Accuracy unit:

[0115] μg / mL

[0116]

[0117] Calibrator

[0118] Accuracy unit: μg / mL

[0119]

[0120] Uniformity unit: μg / mL

[0121] Calibrator 1

[0122]

[0123]

[0124] Uniformity

[0125] Calibrator 2 units: μg / mL

[0126]

[0127] Uniformity

[0128] Calibrator 3 units: μg / mL

[0129]

[0130]

[0131] Quality control products

[0132]

[0133]

[0134] Uniformity

[0135] Quality control sample 1 unit: μg / mL

[0136]

[0137]

[0138] Quality control sample, 2 units: μg / mL

[0139]

[0140] Quality control sample 3 units: μg / mL

[0141]

[0142]

[0143] The above description is merely an embodiment of the present invention and does not limit the scope of protection of the present invention. Any equivalent structural or procedural transformations made using the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A vancomycin derivative, characterized in that, The structural formula of the vancomycin derivative is shown in Formula (Ⅰ): In the formula, R1 is a linking group, and the linking group is -CO-(CH2). n -COOH, -(CH2) n -COOH, -O-(CH2) n -COOH, -S-(CH2) n -COOH, -NH-(CH2) n -COOH, -CS-(CH2) n -COOH, -(CH2) n -NH-COOH or One of them; Preferably, the linking group is -CO-(CH2). n -COOH; More preferably, the linking group is -CO-(CH2)3-COOH; Where n is any integer between 1 and 10.

2. A method for preparing a vancomycin derivative with the structural formula shown in formula (I), characterized in that, The compound is prepared by reacting compound 1 with compound R1-X, which can introduce a linking group R1, and includes the following steps: Compound 1, compound R1-X, and triethylamine were dissolved in dimethylformamide. The solution was stirred at room temperature, and the resulting reaction product was purified. Preferably, high performance liquid chromatography is used for concentration and purification to obtain the vancomycin derivative shown in formula (I), wherein the compound R1-X is a corresponding organic acid or acid anhydride capable of introducing a linking group R1; In the above method, more preferably, compound 1, compound R1-X and triethylamine are added sequentially to dimethylformamide for dissolution, wherein the molar ratio of compound 1 to compound R1-X is 1:1 to 1.5, preferably 1:1.1; the molar ratio of compound 1 to triethylamine is 1:2 to 2.

5. This solution is stirred at room temperature for 10 to 12 hours, and the resulting reaction product is concentrated and purified by high performance liquid chromatography to obtain the vancomycin derivative shown in formula (I). More preferably, the structural formula of the vancomycin derivative is shown in formula (II): More preferably, the method for preparing the vancomycin derivative shown in formula (II) includes the following steps: Compound 1, glutaric anhydride and triethylamine were added to dimethylformamide and dissolved. The solution was stirred at room temperature. After the reaction was completed, the reaction product was purified to obtain the vancomycin derivative shown in formula (II). Preferably, after the reaction is complete, the reaction product obtained is concentrated and purified by high performance liquid chromatography; More preferably, the molar ratio of compound 1 to glutaric anhydride is 1:1 to 1.5, more preferably 1:1.1; and the molar ratio of compound 1 to triethylamine is 1:2 to 2.

5.

3. Vancomycin artificial antigen, characterized in that, The vancomycin derivative of claim 1 is linked to a carrier, and its structural formula is shown in formula (Ⅲ): Wherein, R2 is a linking group, and the linking group is -CO-(CH2). n -CO-, -(CH2) n -CO-, -O-(CH2) n -CO-, -S-(CH2) n -CO-, -NH-(CH2) n -CO-, -CS-(CH2) n -CO-, -(CH2) n -NH-CO- or One of them; Preferably, the linking group is -CO-(CH2). n -CO-; More preferably, the linking group is -CO-(CH2)3-CO-; Where n is any integer between 1 and 10; The carrier is one of serum albumin, immunoglobulin, fibrinogen, ovalbumin, thyroglobulin, hemocyanin, and polylysine, preferably a serum protein, and more preferably bovine serum albumin.

4. A vancomycin-specific antibody, characterized in that, It is obtained by immunizing experimental animals with the vancomycin artificial antigen as described in claim 3 and then isolating and purifying them.

5. The anti-vancomycin specific antibody according to claim 4, characterized in that: The antibody is a polyclonal antibody obtained by boosting the immunization of experimental animals with the vancomycin artificial antigen as described in claim 3; Alternatively, it may be a monoclonal antibody obtained by somatic cell hybridization after immunizing experimental animals with the vancomycin artificial antigen as described in claim 3. Preferably, the host animal is one of rabbit, goat, sheep, rat, mouse, guinea pig or horse.

6. A vancomycin chemiluminescent immunoassay reagent, characterized in that, It contains a vancomycin-specific antibody and an indicator reagent; the indicator reagent is selected from chemiluminescent reagents, enzyme reagents, radioisotope reagents, and fluorescent reagents; preferably, the indicator reagent is a chemiluminescent reagent, composed of a vancomycin-alkaline phosphatase conjugate and a luminescent substrate, wherein the luminescent substrate is 2-chloro-5-(4-methoxyspiro-1,2-dioxane-3,2′-(5-chlorotricyclo3.3.1.13.7decane)-4-yl-1-phenylphosphate disodium salt.

7. A method for preparing the vancomycin chemiluminescent immunoassay reagent according to claim 6, characterized in that, Includes the following steps: Preparation of S1.1 Chemiluminescent reagent 1: Magnetic microparticles coated with anti-vancomycin specific antibody were added to the magnetic bead base solution until the final concentration of the magnetic microparticles coated with anti-vancomycin specific antibody was 0.1 mg / mL. The mixture was vortexed for 1 minute. The pH of the solution was adjusted to 7.10-7.30 with 6N HCl or 3M NaOH. The volume was brought up to 1L with the magnetic bead base solution. After the volume was brought up, the mixture was stirred for another 10 minutes. Preparation of S1.2 Chemiluminescent reagent 2: Add vancomycin-alkaline phosphatase conjugate to alkaline phosphatase base solution until the final concentration of vancomycin-alkaline phosphatase conjugate is 1 KU / L. Stir with a magnetic stirrer for 25-35 minutes. Adjust the pH of the solution to 7.50-7.70 with 6N HCl or 3M NaOH. Make up the volume to 1L with alkaline phosphatase base solution. After making up the volume, continue stirring for ten minutes. Preparation of S1.3 Chemiluminescent reagent 3: Add 90% purified water (v / v), 8.47g of 145mmol / L sodium chloride, and 0.5mL of 0.05% preservative Precursor Pryknin 300. Stir and mix for 25-35 minutes. Adjust the pH of the solution to 7.10-7.30 with 6N HCl or 3M NaOH. Make up the volume to 1L. After making up the volume, continue stirring for ten minutes.

8. The method for preparing the vancomycin chemiluminescent immunoassay reagent according to claim 7, characterized in that, The preparation of the anti-vancomycin specific antibody includes the following steps: A. Place the vancomycin derivative of claim 1 in a container, and add dimethylamide, ethanol, potassium phosphate buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxythiosuccinimide in sequence, and stir to dissolve; B. Dissolve bovine serum albumin in phosphate buffer; C. Add the activated vancomycin derivative from step A to the bovine serum albumin solution obtained in step B, stir at 2-8°C for 8-24 hours, and dialyze the resulting solution using a dialysis bag to obtain vancomycin artificial antigen. D. Dilute the vancomycin artificial antigen synthesized in step C with phosphate buffer, mix the diluted vancomycin artificial antigen solution with Freund's complete adjuvant, and inject it into experimental animals. 14-28 days later, mix the same vancomycin artificial antigen solution with Freund's incomplete adjuvant and inject it into the experimental animals once more. Thereafter, inject once every 14-28 days. After 3-6 months from the first injection, collect blood from the experimental animals, separate and purify the antibodies.

9. The method for preparing the vancomycin chemiluminescent immunoassay reagent according to claim 7, characterized in that, The preparation method of the magnetic microparticles coated with anti-vancomycin specific antibody is as follows: 10 mL of 5 mg / mL magnetic beads, 90 mL of activation solution, 20 μg / mg of anti-vancomycin specific antibody, 500 mg / mL of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 500 mg / mL of N-hydroxythiosuccinimide sodium salt are placed in a rotary incubator and mixed by rotation. The magnetic beads are coated for 120 minutes. The coated magnetic beads are washed three times with washing solution. 100 mL of magnetic bead blocking solution is added and mixed by rotation. The magnetic beads are blocked for 60 minutes. The blocked magnetic beads are washed three times with washing solution. 100 mL of magnetic bead base solution is added to the washed magnetic beads to preserve them. The magnetic bead blocking solution is prepared as follows: 90% purified water (volume fraction), 20.0g of 2% bovine serum albumin, 0.5mL of 0.05% Tween-20, 0.5mL of 0.05% preservative Precambriconazole 300, and 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.10–7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes. The cleaning solution is prepared as follows: 90% purified water (volume fraction), 8.5g of 145mmol / L sodium chloride, 0.95g of 6.66mmol / L disodium hydrogen phosphate, 0.45g of 3.33mmol / L potassium dihydrogen phosphate, 0.5mL of 0.05% preservative Precinctine 300, and 0.5mL of 0.05% Tween-20 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.10-7.30 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes. The preparation method of the magnetic bead base solution is as follows: 90% purified water, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, 2.5g of 0.25% bovine serum albumin, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 6.90-7.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes. The vancomycin-alkaline phosphatase conjugate is prepared as follows: 5.6 μL of 1 KU / mL alkaline phosphatase is added to 0.5 mL of alkaline phosphatase basal solution to prepare an alkaline phosphatase solution. 2 mg of 2 mg / mL vancomycin derivative, 0.5 mL of activation solution, 2 mg / mL of 2 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 2 mg / mL of 2 mg / mL N-hydroxythiosuccinimide sodium salt are placed in a magnetic stirrer and stirred for 5 minutes to activate. The activated vancomycin derivative is added to the above alkaline phosphatase solution and coupled at 2-8℃ for 120 minutes. The coupled vancomycin-alkaline phosphatase conjugate is dialyzed 4 times with dialysis buffer, each dialyzing for 8 hours. The alkaline phosphatase base solution is prepared as follows: 90% purified water (v / v), 1.25g of 0.125% bovine serum albumin, 0.20g of 1mmol / L magnesium chloride, 13.63mg of 0.1mmol / L zinc chloride, 8.5g of 145.5mmol / L sodium chloride, 6.057g of 50mmol / L tris(hydroxymethyl)aminomethane, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25–35 minutes. The pH of the solution is adjusted to 7.50–7.70 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After bringing the volume to 1L, stirring is continued for ten minutes. The preparation method of the activation solution is as follows: 90% purified water (volume fraction), 19.52g of 100mmol / L morpholine ethanesulfonic acid, 0.5mL of 0.05% Tween-20, and 0.5mL of 0.05% preservative Precambriconazole 300 are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 4.90-5.10 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes. The preparation method of the dialysate is as follows: 90% purified water, 8.47g of 145mmol / L sodium chloride, 1.15g of 8.1mmol / L disodium hydrogen phosphate, 0.2g of 1.47mmol / L potassium dihydrogen phosphate, and 0.5mL of 0.05% preservative are stirred and mixed for 25-35 minutes. The pH of the solution is adjusted to 7.30-7.50 with 6N HCl or 3M NaOH, and the volume is brought to 1L. After the volume is brought to 1L, stirring is continued for ten minutes.

10. The detection method of the vancomycin chemiluminescent immunoassay reagent according to claim 6, characterized in that: The vancomycin chemiluminescent immunoassay reagent is assembled into a kit product, which is used to determine the vancomycin content in biological samples. The biological samples are liquid physiological samples of humans or animals, such as serum, plasma, whole blood, urine, saliva, lymph, tissue fluid, cerebrospinal fluid, amniotic fluid, or tears.

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