Olanzapine chemiluminescence immunoassay kit and preparation method thereof

Abstract

The invention relates to the field of preparation of chemiluminescence immunoassay kits, in particular to an olanzapine chemiluminescence immunoassay kit and a preparation method thereof. By introducing innovative components such as an olanzapine metabolic simulant, a nano-enzyme catalysis system and a molecular imprinting interior label, a full-process exclusive optimization system from antibody preservation to signal amplification is constructed, and the industrial problems that a traditional kit is poor in stability, insufficient in specificity and serious in matrix interference are solved. The method has remarkable technical advancement and clinical application value.

Description

Technical Field

[0001] This invention relates to the field of chemiluminescent immunoassay kit preparation, specifically to an olanzapine chemiluminescent immunoassay kit and its preparation method. Background Technology

[0002] Olanzapine has neuroprotective effects, effectively alleviating positive and negative symptoms as well as affective symptoms of schizophrenia, improving cognitive function in patients with schizophrenia, and can also be used for maintenance therapy of schizophrenia to prevent relapse. It rarely causes extrapyramidal reactions, and therefore is widely used clinically. The target steady-state trough concentration range for olanzapine is 20–80 ng / mL.

[0003] Studies using ROC curve analysis have shown a clear correlation between olanzapine blood concentration and clinical efficacy, with the lowest effective blood concentration being 9 ng / ml. Some studies have also found that efficacy is more significant at blood concentrations ≥23.2 ng / ml, suggesting that higher doses of olanzapine may be more effective. However, toxic reactions should be monitored when blood concentrations exceed 100 ng / mL. Generally, olanzapine blood concentration is positively correlated with dose; that is, as the dose of olanzapine increases, the blood concentration will increase accordingly. However, the pharmacokinetics of olanzapine vary considerably among individuals, so continuous monitoring of blood concentration is necessary. Currently, the main methods for detecting olanzapine include liquid chromatography-tandem mass spectrometry (LC-MS / MS), two-dimensional liquid chromatography (2D-LC-UV), and enzyme-linked immunosorbent assay (ELISA). LC-MS / MS involves complex sample pretreatment and procedures, and the expensive instruments make it unsuitable for large-sample testing and for use at the grassroots level. ELISA has lower sensitivity and specificity.

[0004] Therefore, it is essential to invent a method that can quickly and accurately detect olanzapine. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention aims to develop an olanzapine chemiluminescent immunoassay kit and its preparation method. By introducing innovative components such as olanzapine metabolite mimics, nanozyme catalytic systems, and molecularly imprinted internal standards, a dedicated optimized system for the entire process from antibody preservation to signal amplification is constructed. This solves the shortcomings of traditional kits, such as poor stability, insufficient specificity, and severe matrix interference, and has significant technological advancements and clinical application value.

[0006] This invention discloses a method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add activation buffer and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture. After activating the mixture, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody to suspend. After the suspension is completed, aspirate the liquid and add magnetic bead preservation buffer to continue the suspension. Then obtain magnetic beads coated with olanzapine antibody. Then mix the magnetic beads coated with olanzapine antibody with magnetic bead preservation buffer to prepare R1 reagent. (2) Preparation of R2 reagent: Olanzapine synthetic antigen was mixed with alkaline phosphatase and glutaraldehyde was added for a reaction. After the reaction was completed, dialysis was performed to remove unreacted glutaraldehyde and impurities. After dialysis, glycerol was added to obtain enzyme-labeled antigen, and then diluted with enzyme working solution to obtain R2 reagent. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0007] Preferably, in step (1) R1 reagent preparation, the activation buffer is a 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 15-100mM and a pH of 4-6.

[0008] Preferably, in the preparation of reagent R1 in step (1), the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:(10~50).

[0009] Preferably, in step (1) R1 reagent preparation, the mass ratio of the hydroxyl magnetic beads to olanzapine antibody is 1:(2~20).

[0010] Preferably, in step (1) R1 reagent preparation, the magnetic bead preservation buffer is composed of Tris buffer, glycine bovine serum albumin, sodium dodecyl sulfate, surfactant Tween-20, preservative Proclin 300, olanzapine metabolite mimic, zinc finger protein mimic peptide and deep eutectic solvent.

[0011] Preferably, the olanzapine metabolite mimic is 4-amino-2-chloropyridine-N-methylpiperazine; the zinc finger protein mimic peptide has the sequence Cys-His-Glu-Lys-Cys and contains Zn. 2+ Chelating group.

[0012] Preferably, in the sample loading, photographing, and gel cutting steps of S3, the pre-electrophoresis time is 15-30 min; the volume ratio of glycerol to ethylenediaminetetraacetic acid is 1:(0.95-1.05); the concentration of ethylenediaminetetraacetic acid is 0.09-0.11 mol / L; after the sample enters the wells of the polyacrylamide gel double-layer glass plate, the electrophoresis voltage is set to 195-205V, and after the sample exits the wells by electrophoresis, the electrophoresis voltage is adjusted to 115-125V.

[0013] Preferably, the deep eutectic solvent is composed of choline chloride and urea, with a molar ratio of choline chloride to urea of ​​1:2.

[0014] Preferably, in step (2) R2 reagent preparation, the mass ratio of the olanzapine synthetic antigen to alkaline phosphatase is 1:(1~10).

[0015] Preferably, in step (2) R2 reagent preparation, the enzyme working solution includes Tris buffer, preservative Proclin 300, mouse IgG, magnesium chloride, zinc chloride, PEG4000, and ionic liquid-surfactant composite system; the ionic liquid-surfactant composite system is composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a mass ratio of 1:3.

[0016] An olanzapine chemiluminescent immunoassay kit, prepared by any of the olanzapine chemiluminescent immunoassay kit preparation methods described above.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses an olanzapine chemiluminescent immunoassay kit and its preparation method, which has the following characteristics: (1) During the preparation of reagent R1, 4-amino-2-chloropyridine-N-methylpiperazine, a mimic of olanzapine metabolites, was added as a conformational stabilizer. 4-amino-2-chloropyridine-N-methylpiperazine has a structure similar to the main metabolite of olanzapine and can competitively bind to the antigen-binding sites of antibodies on the surface of magnetic beads, maintaining the native conformation of the antibody. A zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn) was introduced. 2+ Chelating groups act as dispersants for magnetic beads, chelating free Zn on the surface of the magnetic beads. 2+ This process blocks non-specific metal bridging between magnetic beads, maintaining their monodisperse state. It also synergistically reduces aggregation tendency in conjunction with traditional surfactants. The addition of choline chloride-urea deep eutectic solvent acts as a protective agent, stabilizing the antibody's tertiary structure through a hydrogen bond network. This inhibits protein denaturation during storage, preventing loss of antibody activity.

[0018] (2) During the preparation of reagent R2, an ionic liquid-surfactant composite system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a mass ratio of 1:3 is added. The ionic liquid penetrates into the hydrophobic core of the blood sample protein and works with the surfactant to dissociate the olanzapine-protein complex, releasing free olanzapine molecules.

[0019] (3) The olanzapine chemiluminescent immunoassay kit prepared by the present invention not only significantly improves the specificity of olanzapine detection, but also enhances the stability, anti-interference ability and detection limit of the kit. The one-step sample addition shortens the detection time and meets the high-throughput clinical needs. Attached Figure Description

[0020] Figure 1 Main curve chart Detailed Implementation The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0021] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0022] Example 1: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 15mM and a pH of 4, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:10. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:2. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare 70 mM Tris buffer and adjust the pH to 6 as the base for the magnetic bead storage buffer. Then add 0.05% glycine, 0.1% bovine serum albumin (BSA), 0.5% sodium dodecyl sulfate (SDS), 0.02% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.05% Tween-20, 0.01% Proclin 300, and 0.01% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 5% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 1 mg / mL olanzapine antibody. These beads were then diluted with the magnetic bead preservation buffer to a concentration of 0.1 mg / mL as R1 reagent.

[0023] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 0.5 mg / mL. Prepare a 70 mM Tris buffer and adjust the pH to 6 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 0.05 μg / ml mouse IgG, 20 μM magnesium chloride, 10 μM zinc chloride, 0.1% PEG4000, 0.05% Tween 20, and 0.01% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 0.5 mg / mL to 0.1 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0024] Example 2: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 30mM 2-(n-morpholine) ethanesulfonic acid buffer with pH 4.5 and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:10. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:2. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 60mM Tris buffer solution and adjust the pH to 6.5 as the base for the magnetic bead storage buffer. Then add 0.06% glycine, 0.8% bovine serum albumin (BSA), 1.2% sodium dodecyl sulfate (SDS), 0.08% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.08% Tween-20, 0.03% Proclin 300, and 0.03% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 8% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 1 mg / mL olanzapine antibody. These beads were then diluted with the magnetic bead preservation buffer to a concentration of 0.1 mg / mL as R1 reagent.

[0025] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 0.5 mg / mL. Prepare a 60 mM Tris buffer and adjust the pH to 6.5 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 0.12 μg / ml mouse IgG, 50 μM magnesium chloride, 30 μM zinc chloride, 0.8% PEG4000, 0.08% Tween 20, and 0.03% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 0.5 mg / mL to 0.1 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0026] Example 3: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 50 mM and a pH of 6, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:10. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:2. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 50mM Tris buffer, adjust the pH to 7, and use it as the base for the magnetic bead storage buffer. Then add 1.0% glycine, 2.0% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), 0.13% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.1% Tween-20, 0.1% Proclin 300, and 0.06% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 10% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with olanzapine antibody at a concentration of 1 mg / mL. This was then diluted with the magnetic bead preservation buffer to a concentration of 0.1 mg / mL as R1 reagent.

[0027] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 0.5 mg / mL. Prepare a 50 mM Tris buffer and adjust the pH to 7 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 1.0 μg / ml mouse IgG, 40 μM magnesium chloride, 20 μM zinc chloride, 2.0% PEG4000, 0.1% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 0.5 mg / mL to 0.1 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0028] Example 4: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 70mM 2-(n-morpholine) ethanesulfonic acid buffer with pH 5 and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:10. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:2. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 70mM Tris buffer solution and adjust the pH to 7.5 as the base for the magnetic bead storage buffer. Then add 0.07% glycine, 3.0% bovine serum albumin (BSA), 3.0% sodium dodecyl sulfate (SDS), 0.15% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.08% Tween-20, 0.03% Proclin 300, and 0.08% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 12% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with olanzapine antibody at a concentration of 1 mg / mL. This was then diluted with the magnetic bead preservation buffer to a concentration of 0.1 mg / mL as R1 reagent.

[0029] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 0.5 mg / mL. Prepare a 70 mM Tris buffer and adjust the pH to 7.5 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 3.0 μg / ml mouse IgG, 80 μM magnesium chloride, 40 μM zinc chloride, 3.0% PEG4000, 0.15% Tween 20, and 0.08% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 0.5 mg / mL to 0.1 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0030] Example 5: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 100mM 2-(n-morpholine) ethanesulfonic acid buffer with pH 5.5 and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:10. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:2. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare 100mM Tris buffer and adjust the pH to 8 as the base for the magnetic bead storage buffer. Then add 0.09% glycine, 5.0% bovine serum albumin (BSA), 5.0% sodium dodecyl sulfate (SDS), 0.2% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.2% Tween-20, 0.06% Proclin 300, and 0.1% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 15% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with olanzapine antibody at a concentration of 1 mg / mL. This was then diluted with the magnetic bead preservation buffer to a concentration of 0.1 mg / mL as R1 reagent.

[0031] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 0.5 mg / mL. Prepare a 100mM Tris buffer and adjust the pH to 8 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 5.0 μg / ml mouse IgG, 100 μM magnesium chloride, 50 μM zinc chloride, 5.0% PEG4000, 0.2% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 0.5 mg / mL to 0.1 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0032] Example 6: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 50 mM and a pH of 6, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:20. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:8. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 50mM Tris buffer, adjust the pH to 7, and use it as the base for the magnetic bead storage buffer. Then add 1.0% glycine, 2.0% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), 0.13% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.1% Tween-20, 0.1% Proclin 300, and 0.06% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 10% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 3 mg / mL olanzapine antibody. This was then diluted with the magnetic bead preservation buffer to a concentration of 0.3 mg / mL as R1 reagent.

[0033] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 1.0 mg / mL. Prepare a 50 mM Tris buffer and adjust the pH to 7 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 1.0 μg / ml mouse IgG, 40 μM magnesium chloride, 20 μM zinc chloride, 2.0% PEG4000, 0.1% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 1.0 mg / mL to 0.3 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0034] Example 7: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 50 mM and a pH of 6, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:30. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:12. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 50mM Tris buffer, adjust the pH to 7, and use it as the base for the magnetic bead storage buffer. Then add 1.0% glycine, 2.0% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), 0.13% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.1% Tween-20, 0.1% Proclin 300, and 0.06% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 10% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 5 mg / mL olanzapine antibody. These beads were then diluted with the magnetic bead preservation buffer to a concentration of 0.5 mg / mL as R1 reagent.

[0035] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 1.5 mg / mL. Prepare a 50 mM Tris buffer and adjust the pH to 7 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 1.0 μg / ml mouse IgG, 40 μM magnesium chloride, 20 μM zinc chloride, 2.0% PEG4000, 0.1% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 1.5 mg / mL to 0.6 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0036] Example 8: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 50 mM and a pH of 6, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:40. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:15. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 50mM Tris buffer, adjust the pH to 7, and use it as the base for the magnetic bead storage buffer. Then add 1.0% glycine, 2.0% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), 0.13% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.1% Tween-20, 0.1% Proclin 300, and 0.06% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+ A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 10% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 7 mg / mL olanzapine antibody. These beads were then diluted with the magnetic bead preservation buffer to a concentration of 0.7 mg / mL as R1 reagent.

[0037] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 1.7 mg / mL. Prepare a 50 mM Tris buffer and adjust the pH to 7 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 1.0 μg / ml mouse IgG, 40 μM magnesium chloride, 20 μM zinc chloride, 2.0% PEG4000, 0.1% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 1.7 mg / mL to 0.8 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0038] Example 9: A method for preparing an olanzapine chemiluminescent immunoassay kit, comprising the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 50 mM and a pH of 6, and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture, wherein the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:50. After activating the mixture for 30 min, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody and suspend for 4 h, wherein the mass ratio of hydroxyl magnetic beads to olanzapine antibody is 1:20. After mixing evenly, aspirate the liquid after suspension and leave the solid for later use. Prepare a 50mM Tris buffer, adjust the pH to 7, and use it as the base for the magnetic bead storage buffer. Then add 1.0% glycine, 2.0% bovine serum albumin (BSA), 1.0% sodium dodecyl sulfate (SDS), 0.13% olanzapine metabolite mimic (4-amino-2-chloropyridin-N-methylpiperazine), 0.1% Tween-20, 0.1% Proclin 300, and 0.06% zinc finger protein mimic peptide (sequence: Cys-His-Glu-Lys-Cys, containing Zn). 2+A magnetic bead preservation buffer was prepared by uniformly mixing chelating groups and 10% choline chloride-urea deep eutectic solvent, wherein the molar ratio of choline chloride to urea was 1:2. The solid residue after aspirating the liquid was then added to the prepared magnetic bead preservation buffer and resuspended for 3 hours to obtain magnetic beads coated with 10 mg / mL olanzapine antibody. These beads were then diluted with the magnetic bead preservation buffer to a concentration of 1.0 mg / mL as R1 reagent.

[0039] (2) Preparation of R2 reagent: Olanzapine synthetic antigen and alkaline phosphatase were mixed at a mass ratio of 1:1, and glutaraldehyde was added for a reaction. After the reaction was completed, the mixture was dialyzed with TBS (Tris buffered saline) to remove unreacted glutaraldehyde and other small molecule impurities. After dialyzing, an equal amount of glycerol was added and mixed to obtain enzyme-labeled antigen with a protein concentration of 2.0 mg / mL. Prepare a 50 mM Tris buffer and adjust the pH to 7 as the base buffer for the enzyme working solution. Add an ionic liquid-surfactant complex system composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a 1:3 mass ratio to the base buffer. Then add 1.0 μg / ml mouse IgG, 40 μM magnesium chloride, 20 μM zinc chloride, 2.0% PEG4000, 0.1% Tween 20, and 0.1% Proclin 300 to prepare the enzyme working solution. Then use the enzyme working solution to dilute the enzyme-labeled antigen with a protein concentration of 2.0 mg / mL to 1.0 mg / mL to obtain reagent R2. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

[0040] Example 10: In step (1) R1 reagent preparation, olanzapine metabolite mimic (4-amino-2-chloropyridine-N-methylpiperazine) was not added to the magnetic bead storage buffer, and the proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0041] Example 11: In step (1) during the preparation of reagent R1, no zinc finger protein mimic peptide was added to the magnetic bead preservation buffer, and the proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0042] Example 12: In step (1) during the preparation of reagent R1, no choline chloride-urea deep eutectic solvent was added to the magnetic bead preservation buffer, and the proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0043] Example 13: In step (1) R1 reagent preparation, olanzapine metabolite mimic (4-amino-2-chloropyridine-N-methylpiperazine), zinc finger protein mimic peptide, and choline chloride-urea deep co-crystallization solvent were not added to the magnetic bead preservation buffer. The proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0044] Example 14: In step (2) during the preparation of reagent R2, no ionic liquid-surfactant composite system was added to the magnetic bead storage buffer, and the proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0045] Example 15: In step (1) R1 reagent preparation, olanzapine metabolite mimic (4-amino-2-chloropyridine-N-methylpiperazine), zinc finger protein mimic peptide, and choline chloride-urea deep co-crystallization solvent were not added to the magnetic bead preservation buffer; the proportions of the remaining components were the same as in Example 6. In step (2) R2 reagent preparation, ionic liquid-surfactant composite system was not added to the magnetic bead preservation buffer; the proportions of the remaining components were the same as in Example 6. The remaining steps were the same as in Example 6.

[0046] The stability, anti-interference ability, detection limit, and detection time of the olanzapine chemiluminescent immunoassay kits prepared in Examples 1-13 were tested, and the results are shown in Table 1. Table 1

[0047] Examples 1-9 screened the pH value of the reagents, the proportion of each component parameter in the magnetic bead preservation buffer and the enzyme working solution, while Examples 10-15 screened the components in the magnetic bead preservation buffer and the enzyme working solution.

[0048] The data from Examples 6 and 12 in the table above show that Example 6 (30 days) performed best, significantly outperforming the other examples, while Example 12 (without deep eutectic solvent) showed the worst stability (12 days), demonstrating that this component is crucial for long-term stability. This is because the addition of glycine, bovine serum albumin, and SDS during the sealing process of carboxylated magnetic beads can enhance their stability. The choline chloride-urea deep eutectic solvent stabilizes the antibody and enzyme structures on the surface of the magnetic beads through a hydrogen bond network and works synergistically with the neutral pH (7.0) Tris buffer to reduce protein denaturation, further extending the shelf life.

[0049] Data from Examples 6, 10, and 11 in the table above show that Example 6 (5.2% deviation) exhibits the strongest anti-interference ability and highest sensitivity. Examples 10 (no metabolite mimicry), 11 (no zinc finger protein mimicry peptide), and 14 (no ionic liquid-surfactant complex system) show significantly increased deviations, with the detection limit of Example 14 (no ionic liquid-surfactant complex system) rising to 4.3 ng / mL. This is because the zinc finger protein mimicry peptide (Cys-His-Glu-Lys-Cys) chelates metal ions (such as Zn). 2+ This reduces non-specific adsorption; olanzapine metabolite mimics (4-amino-2-chloropyridine-N-methylpiperazine) competitively bind to interfering molecules, reducing cross-reactivity; when mouse IgG, MgCl2, ZnCl2, and PEG4000 are used in combination, they can ensure the activity of alkaline phosphatase while eliminating non-specific binding in olanzapine detection and reducing the binding of olanzapine to proteins in blood samples; through its ionic liquid and surfactant properties, it penetrates into the hydrophobic core of blood sample proteins, synergistically dissociating olanzapine from the binding of proteins in blood samples with surfactants, while reducing background noise and improving the signal-to-noise ratio, thereby improving the accuracy of detection.

[0050] The data in the table above shows that Example 6 (30 min) had the shortest detection time. The detection times for Examples 10-15 were extended to 45-60 min, indicating that the olanzapine chemiluminescent immunoassay kit prepared by the method provided in this invention reacts rapidly and can effectively reduce detection time.

[0051] Example 15 lacks a deep eutectic solvent, olanzapine metabolite mimics, zinc finger protein mimic peptides, and an ionic liquid-surfactant complex system. Its stability is only 6 days, with a deviation as high as 35.2%, a detection limit of 5.0 ng / mL, and a detection time as long as 60 min. This indicates that the lack of multiple components leads to uncontrolled adsorption of interfering substances, resulting in signal attenuation and increased noise, a significant decrease in reaction rate, and an additive effect.

[0052] A master curve was established using olanzapine manufacturer's reference standards at concentrations of 0 mg / L (negative sample), 6.124 mg / L, 9.748 mg / L, 21.245 mg / L, 28.142 mg / L, 42.784 mg / L, 65.124 mg / L, 78.145 mg / L, and 101.23 mg / L. The values ​​of each manufacturer's reference standard were determined by mass spectrometry. The master curve is shown below. Figure 1 As shown in Table 2: Table 2

[0053] The olanzapine chemiluminescent immunoassay kit prepared in Example 6 of this invention was compared with liquid chromatography-tandem mass spectrometry (LC-MS / MS). Simultaneously, blood samples with concentrations ranging from 6.0 mg / L to 100 mg / L were detected. The deviation between the olanzapine chemiluminescent immunoassay kit and the concentration detected by LC-MS / MS was calculated and recorded, as shown in Table 3. Table 3

[0054] As shown in Table 3, the olanzapine chemiluminescent immunoassay kit prepared in this invention exhibits a deviation of no more than 10% from the detection results of liquid chromatography-tandem mass spectrometry in the detection of low, medium, and high concentration samples. This further demonstrates that the olanzapine chemiluminescent immunoassay kit prepared in this invention has excellent stability and accuracy.

[0055] In summary, this invention achieves comprehensive optimization of stability, anti-interference ability, sensitivity, and detection speed by optimizing the magnetic bead coating concentration, enzyme-labeled antigen concentration, and the synergistic effect of key additives (deep eutectic solvent, zinc finger protein mimic peptide, metabolite mimic, and ionic liquid-surfactant composite system).

[0056] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing an olanzapine chemiluminescent immunoassay kit, characterized in that, Includes the following steps: (1) Preparation of R1 reagent: Place the magnetic beads in a shaker, add activation buffer and shake to remove impurities on the surface of the magnetic beads. Then add 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide to form a mixture. After activating the mixture, aspirate the liquid in the mixture to obtain hydroxyl magnetic beads. Then add olanzapine antibody to suspend. After the suspension is completed, aspirate the liquid and add magnetic bead preservation buffer to continue the suspension. Then obtain magnetic beads coated with olanzapine antibody. Then mix the magnetic beads coated with olanzapine antibody with magnetic bead preservation buffer to prepare R1 reagent. (2) Preparation of R2 reagent: Olanzapine synthetic antigen was mixed with alkaline phosphatase and glutaraldehyde was added for a reaction. After the reaction was completed, dialysis was performed to remove unreacted glutaraldehyde and impurities. After dialysis, glycerol was added to obtain enzyme-labeled antigen, and then diluted with enzyme working solution to obtain R2 reagent. (3) Preparation of olanzapine chemiluminescent immunoassay kit: The R1 reagent prepared in step (1) is combined with the R2 reagent prepared in step (2) to obtain the olanzapine chemiluminescent immunoassay kit.

2. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1) R1 reagent preparation, the activation buffer is a 2-(n-morpholine) ethanesulfonic acid buffer with a concentration of 15~100mM and a pH of 4~6.

3. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1) R1 reagent preparation, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbonyl and N-hydroxysuccinimide is 1:(10~50).

4. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1) R1 reagent preparation, the mass ratio of the hydroxy magnetic beads to olanzapine antibody is 1:(2~20).

5. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (1) R1 reagent preparation, the magnetic bead preservation buffer is composed of Tris buffer, glycine bovine serum albumin, sodium dodecyl sulfate, surfactant Tween-20, preservative Proclin 300, olanzapine metabolite mimic, zinc finger protein mimic peptide and deep cocrystallization solvent.

6. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 5, characterized in that, The olanzapine metabolite mimic is 4-amino-2-chloropyridine-N-methylpiperazine; the zinc finger protein mimic peptide has the sequence Cys-His-Glu-Lys-Cys and contains Zn. 2+ Chelating group.

7. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 5, characterized in that, The deep eutectic solvent is composed of choline chloride and urea, with a molar ratio of choline chloride to urea of ​​1:

2.

8. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (2) R2 reagent preparation, the mass ratio of the olanzapine synthetic antigen to alkaline phosphatase is 1:(1~10).

9. The method for preparing an olanzapine chemiluminescent immunoassay kit according to claim 1, characterized in that, In step (2) R2 reagent preparation, the enzyme working solution includes Tris buffer, preservative Proclin 300, mouse IgG, magnesium chloride, zinc chloride, PEG4000, and ionic liquid-surfactant composite system; the ionic liquid-surfactant composite system is composed of 1-butyl-3-methylimidazolium hexafluorophosphate and polyoxyethylene lauryl ether in a mass ratio of 1:

3.

10. An olanzapine chemiluminescent immunoassay kit prepared by the method described in any one of claims 1 to 9.

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