Detection kit for valproic acid and application thereof

Abstract

The invention discloses a valproic acid detection kit and application thereof.The valproic acid detection kit comprises a reagent R1 and a reagent R2, the reagent R1 is a carboxyl magnetic bead reagent coated with a valproic acid antibody, and the concentration of carboxyl magnetic beads is 0.1-1 mg / mL; the R2 reagent is a valproic acid synthetic antigen-alkaline phosphatase marker reagent, and the protein concentration of the R2 reagent is 0.1-2 mg / ml. The valproic acid detection kit is composed of specific reagents, the storage stability is improved, non-specific binding is reduced, a detection value stably falls within an acceptable error range of a true value, the reliability of a detection result is improved, meanwhile, expensive instruments are not needed, the cost is reduced, and the detection kit is suitable for large-scale popularization and application. Meanwhile, valproic acid in a sample can be rapidly, simply, stably and accurately detected.

Description

Technical Field

[0001] This invention relates to a valproic acid detection kit and its application, belonging to the field of in vitro detection technology. Background Technology

[0002] Valproic acid is a broad-spectrum antiepileptic drug, mainly used clinically to treat various types of epileptic seizures, including generalized tonic-clonic seizures, myoclonic seizures, and petit mal seizures. It is frequently used in clinical practice. Valproic acid is also used to treat manic episodes associated with bipolar disorder, migraines, schizophrenia, and other diseases.

[0003] Valproic acid exhibits significant individual differences in pharmacokinetics and pharmacodynamics, making dosage control challenging. If serum or plasma concentrations are too low, epileptic control cannot be maintained; conversely, excessively high concentrations increase the risk of toxic reactions. The drug's half-life is difficult to predict for any given individual; furthermore, studies have shown a positive correlation between valproic acid plasma concentrations and the incidence of adverse drug reactions (ADRs). Therefore, close monitoring of serum or plasma valproic acid concentrations is essential for patients undergoing valproic acid therapy. To maximize efficacy and minimize adverse reactions, therapeutic drug monitoring is necessary during the clinical application of valproic acid.

[0004] Currently, the main methods for detecting valproic acid include liquid chromatography-tandem mass spectrometry (LC-MS), two-dimensional liquid chromatography (2D-LC-UV), and enzyme-linked immunosorbent assay (ELISA). The sample pretreatment for LC-MS is relatively complex, and the operation steps are also relatively complicated. In addition, the cost of LC-MS instruments is high, resulting in high detection costs, which makes it unsuitable for large-sample testing and grassroots implementation. ELISA has low sensitivity and specificity. Summary of the Invention

[0005] In response to at least one problem existing in the prior art, the present invention provides a valproic acid detection kit and its application. The kit uses specific reagents, eliminates the need for expensive instruments, reduces costs, and provides a rapid, simple, stable, and accurate detection method for valproic acid in samples.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a valproic acid detection kit, comprising reagent R1 and reagent R2, wherein reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody, and reagent R2 is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent.

[0007] Preferably, the method for preparing the carboxyl magnetic bead reagent coated with valproic acid antibody includes: washing and activating carboxyl magnetic beads, mixing the activated carboxyl magnetic beads with valproic acid antibody, and reacting at 20-30°C for 3-4 hours to obtain carboxyl magnetic beads coated with valproic acid antibody, blocking, and mixing the carboxyl magnetic beads coated with valproic acid antibody with buffer I to obtain the carboxyl magnetic bead reagent coated with valproic acid antibody.

[0008] Preferably, the buffer I is a Tris buffer containing Proclin 300, glucose, mannitol and EDTA.

[0009] Preferably, the buffer I is a 50-500 mM Tris buffer containing 0.01-0.1% Proclin 300, 0.05-2.0% glucose, 0.1-5% mannitol and 0.5-5% EDTA, with a pH of 6-8.

[0010] Preferably, the mass ratio of the carboxyl magnetic beads to the valproic acid antibody is 50 to 2:1.

[0011] Preferably, in the carboxyl magnetic bead reagent coated with valproic acid antibody, the concentration of carboxyl magnetic beads is 0.1–1 mg / mL.

[0012] Preferably, the carboxyl magnetic bead cleaning and activation treatment includes: taking carboxyl magnetic beads and resuspending them in an activation buffer solution for cleaning, then adding a mixture of EDC and NHS to the cleaned carboxyl magnetic beads and activating them for 20-30 minutes.

[0013] Preferably, the mass ratio of the carboxyl magnetic beads to EDC is 10 to 50:1, and the mass ratio of EDC to NHS is 1:1.

[0014] Preferably, the activation buffer is a 15-100 mM MES buffer with a pH of 4-6.

[0015] Preferably, in the carboxyl magnetic bead reagent coated with valproic acid antibody, the particle size of the carboxyl magnetic beads is 1-3 μm.

[0016] Preferably, the preparation method of the valproic acid synthetic antigen-alkaline phosphatase labeling reagent includes: mixing valproic acid synthetic antigen, alkaline phosphatase, and TBS buffer and dialyzing, then adding a coupling agent and reacting in the dark for 2-3 hours, and after the reaction, adding TBS buffer and dialyzing to obtain valproic acid synthetic antigen-alkaline phosphatase labeling, and mixing the valproic acid synthetic antigen-alkaline phosphatase labeling with buffer II to obtain the valproic acid synthetic antigen-alkaline phosphatase labeling reagent.

[0017] Preferably, the protein concentration in the valproic acid synthesis antigen-alkaline phosphatase labeling reagent is 0.1–2 mg / ml.

[0018] Preferably, the buffer II is a Tris buffer containing Proclin 300, mouse IgG, histidine, glycerol and PEG 600.

[0019] Preferably, the buffer II is a 50-500 mM Tris buffer containing 0.01-0.1% Proclin 300, 0.05-5 μg / mL mouse IgG, 0.05-5% histidine, 10-50% glycerol and 0.1-5% PEG 600, with a pH of 6-8.

[0020] Preferably, the mass ratio of the valproic acid synthetic antigen to alkaline phosphatase is 1:1 to 10.

[0021] Preferably, the coupling agent is 0.05-1% of the total mass of valproic acid synthetic antigen and alkaline phosphatase.

[0022] Preferably, the coupling agent is one of glutaraldehyde, sodium periodate, or N-hydroxysuccinimide maleimide, with glutaraldehyde being the most preferred.

[0023] This invention provides a method for detecting valproic acid content in serum using a chemiluminescent immunoassay kit based on the present invention.

[0024] Compared with existing technologies, the valproic acid detection kit of this invention uses coupled magnetic beads with carboxyl functional groups, which is low in cost; it uses a buffer containing glucose, mannitol, and EDTA to preserve the valproic acid antibody coated on the magnetic beads, which improves the stability during preservation and the reliability of the detection results; it uses a buffer containing mouse IgG, histidine, glycerol, and PEG600 to preserve the alkaline phosphatase-coupled valproic acid synthetic antigen, which can eliminate non-specific binding and reduce the binding of valproic acid to proteins in blood samples, so that the detection value falls stably within the acceptable error range of the true value, thus improving the reliability of the detection results; the valproic acid detection kit of this invention still has good stability and reliability of detection results after being stored at 4°C for 1 year; the application of the valproic acid detection kit of this invention can automatically analyze and detect samples through the closed kit and instrument detection system, reducing systematic errors and eliminating errors introduced by manual operation, resulting in high detection efficiency and broad application prospects. Attached Figure Description

[0025] Figure 1 This is a standard curve diagram of Embodiment 1 of the present invention; Detailed Implementation

[0026] The following is a clear and complete description of the technical solutions in the implementation of this invention. The described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply.

[0027] This invention provides a valproic acid detection kit, comprising reagent R1 and reagent R2, wherein reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody, and the concentration of the carboxyl magnetic bead is 0.1-1 mg / mL, and reagent R2 is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent, and the protein concentration is 0.1-2 mg / mL.

[0028] In this invention, the preparation method of carboxyl magnetic beads coated with valproic acid antibody includes: resuspending and washing carboxyl magnetic beads with activation buffer, then adding a mixture of EDC and NHS to the washed carboxyl magnetic beads, wherein the mass ratio of carboxyl magnetic beads to EDC is 10-50:1 and the mass ratio of EDC to NHS is 1:1, activating for 20-30 min, mixing the activated carboxyl magnetic beads with valproic acid antibody, wherein the mass ratio of carboxyl magnetic beads to valproic acid antibody is 50-2:1, reacting at 20-30°C for 3-4 h, blocking, and obtaining carboxyl magnetic beads coated with valproic acid antibody, and mixing the carboxyl magnetic beads coated with valproic acid antibody with buffer I to obtain carboxyl magnetic bead reagent coated with valproic acid antibody.

[0029] In this invention, buffer I comprises 50-500 mM Tris buffer with a pH of 6-8, and buffer I further comprises Proclin 300, glucose, mannitol and EDTA, wherein Proclin 300 is 0.01-0.1%, glucose is 0.05-2.0%, mannitol is 0.1-5% and EDTA is 0.5-5%.

[0030] In this invention, the activation buffer is a 15-100 mM MES buffer with a pH of 4-6.

[0031] In this invention, the carboxyl magnetic bead reagent coated with valproic acid antibody has a particle size of 1-3 μm, preferably 1 μm, 2 μm, or 3 μm.

[0032] In this invention, the preparation method of the valproic acid synthetic antigen-alkaline phosphatase labeling reagent includes: mixing valproic acid synthetic antigen, alkaline phosphatase, and TBS buffer and dialyzing, wherein the mass ratio of valproic acid synthetic antigen to alkaline phosphatase is 1:1 to 10; then adding a coupling agent and reacting in the dark for 2 to 3 hours, wherein the coupling agent is 0.05 to 1% of the total mass of valproic acid synthetic antigen and alkaline phosphatase; after the reaction, adding TBS buffer and dialyzing to obtain the valproic acid synthetic antigen-alkaline phosphatase labeling; mixing the valproic acid synthetic antigen-alkaline phosphatase labeling with buffer II to obtain the valproic acid synthetic antigen-alkaline phosphatase labeling reagent, wherein the protein concentration (excluding the protein concentration of mouse IgG) is 0.1 to 2 mg / ml.

[0033] In this invention, the buffer II comprises 50-500 mM Tris buffer with a pH of 6-8, and the buffer II further comprises Proclin 300, mouse IgG, histidine, glycerol and PEG600, wherein Proclin 300 is 0.01-0.1%, mouse IgG is 0.05-5 μg / mL, histidine is 0.05-5%, glycerol is 10-50% and PEG600 is 0.1-5%.

[0034] In this invention, the coupling agent is one of glutaraldehyde, sodium periodate, or N-hydroxysuccinimide maleimide, with glutaraldehyde being preferred.

[0035] In this invention, the valproic acid detection kit of this invention is used to apply a chemiluminescent immunoassay method for detecting the valproic acid content in serum, as detailed below:

[0036] (1) Dissolve the standard containing valproic acid in pure water and prepare different concentrations as standard solutions. Add the above-mentioned reagents R1 and R2 to the reaction tube, shake to mix, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the standard solution concentration. Plot a standard curve of standard solution concentration versus luminescence value.

[0037] (2) Add the above-mentioned reagents R1 and R2 to the treated blood to be tested into the reaction tube, shake to mix, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the concentration of the standard solution. Then calculate the valproic acid in the sample according to the standard curve.

[0038] Example 1

[0039] A valproic acid detection kit includes reagent R1 and reagent R2, wherein reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody, the concentration of the carboxyl magnetic bead is 1 mg / mL, and reagent R2 is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent, the protein concentration is 1 mg / mL.

[0040] The carboxyl magnetic bead reagent coated with valproic acid antibody, i.e., reagent R1, is prepared as follows:

[0041] (a1) Reagent preparation: Activation buffer: Prepare a 50mM MES buffer with pH 6 using MES and pure water as raw materials.

[0042] Buffer I: A 50 mM Tris buffer with pH 7 was prepared using Tris·HCl, Proclin 300, glucose, mannitol and EDTA as raw materials, with Proclin 300 0.1%, glucose 1%, mannitol 2% and EDTA 1%.

[0043] EDC solution: Prepare a 10 mg / mL EDC solution using EDC and activation buffer as raw materials;

[0044] NHS solution: A 10 mg / mL NHS solution was prepared using Sulfo-NHS and activation buffer as raw materials.

[0045] EDC and NHS mixture: Mix 10 mg / mL EDC solution with 10 mg / mL NHS solution at a volume ratio of 1:1;

[0046] (a2) Washing and activation of carboxyl magnetic beads: Take 10 mg of carboxyl magnetic beads with a particle size of 1 μm, add them to a 10 mL coated tube, magnetically separate them, and aspirate the supernatant. Then, take 2.5 mL of activation buffer and add it to the coated tube to resuspend the carboxyl magnetic beads. Mix at 2000 rpm for 10 s, magnetically separate them, and aspirate the supernatant. Take 200 μL of EDC and NHS mixture and add it to the coated tube. Mix at 2000 rpm for 10 s, and then place it on a roller at 25℃ for 30 min to activate. Take 2.5 mL of activation buffer and add it to the coated tube to resuspend it. Magnetically separate it, and aspirate the supernatant to obtain activated carboxyl magnetic beads.

[0047] (a3) Carboxyl magnetic beads coated with valproic acid antibody: The activated carboxyl magnetic beads were resuspended in 2.5 mL of activation buffer, 200 μg of valproic acid monoclonal antibody was added, and the mixture was mixed at 2000 rpm for 10 s. The mixture was then reacted on a roller at 25 °C for 4 h. After magnetic separation, the supernatant was collected, and the mixture was resuspended in 2.5 mL of buffer I. The mixture was then blocked on a roller at 25 °C for 2 h. After blocking, the mixture was magnetically separated, and the supernatant was collected to obtain carboxyl magnetic beads coated with valproic acid antibody.

[0048] (a4) Carboxyl magnetic bead reagent coated with valproic acid antibody: Take the above-mentioned carboxyl magnetic beads coated with valproic acid antibody, add buffer I to dilute them so that the concentration of carboxyl magnetic beads is 1 mg / mL, and obtain carboxyl magnetic bead reagent coated with valproic acid antibody.

[0049] The valproic acid-based antigen-alkaline phosphatase labeling reagent, i.e., reagent R2, is prepared as follows:

[0050] (b1) Reagent preparation: Buffer II: Prepare a 50mM Tris buffer with pH 7 using Tris·HCl, Proclin 300, mouse IgG, histidine, glycerol and PEG600 as raw materials, and Proclin 300 0.1%, mouse IgG 1μg / mL, histidine 1%, glycerol 30% and PEG600 2%;

[0051] (b2) Valproic acid synthesis antigen-alkaline phosphatase label: 100 μg of valproic acid synthesis antigen was pipetted into a 2 mL labeling tube, 1.0 mg of alkaline phosphatase was added, and then 1×TBS buffer was added to a total volume of 150 μL. After mixing, the mixture was placed in a dialysis bag, and dialysis was carried out overnight at 2–8 °C with stirring using 1 L of 1×TBS buffer. The dialyzed valproic acid synthesis antigen and alkaline phosphatase were then placed into a 2 mL labeling tube, 10 μg of glutaraldehyde was added, and the mixture was mixed for 10 s. The mixture was then placed in a 25 °C dark environment for 3 h. After dialysis, 1 L of 1×TBS buffer was added, and dialysis was carried out overnight at 2–8 °C with stirring. After dialysis, the valproic acid synthesis antigen-alkaline phosphatase label was obtained. The total protein content of the dialysis product (excluding the protein concentration of mouse IgG) was detected by UV spectrophotometer. The average value was taken from 3 measurements.

[0052] (b3) Valproic acid synthesis antigen-alkaline phosphatase labeling reagent: Based on the total protein content in the above valproic acid synthesis antigen-alkaline phosphatase labeling reagent, add buffer II to dilute it to a protein concentration of 1 mg / mL (excluding the protein concentration of mouse IgG) to obtain the valproic acid synthesis antigen-alkaline phosphatase labeling reagent.

[0053] The method for detecting valproic acid in serum using the above-mentioned valproic acid detection kit is as follows:

[0054] (1) Dissolve the standard containing valproic acid in pure water to prepare different concentrations: 3.035 μg / mL, 8.049 μg / mL, 16.933 μg / mL, 29.609 μg / mL, 043.05 μg / mL, 72.788 μg / mL, 101.24 μg / mL, 132.14 μg / mL, and 149.232 μg / mL, as standard solutions. Add the above-mentioned reagents R1 and R2 to the different standard solutions and place them in reaction tubes. Shake to mix and incubate at 37°C for 15 min. Then, use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the standard solution concentration, as shown in Table 1. Plot a standard curve of standard solution concentration (x) versus luminescence value (y). The standard curve is shown in Table 1. Figure 1 As shown, y = (ad) / (1 + (x / c)) b)+d, where a=40788387.57, b=1.06444597, c=3.23826984, d=-204024.7222.

[0055] Table 1

[0056] Serial Number Concentration (μg / mL) Luminous value 1 3.05 20998069 2 8.049 10833826 3 16.933 6165040 4 29.609 3285695 5 43.05 2207844 6 72.788 1168432 7 101.24 898175 8 132.14 589644 9 149.232 459972

[0057] (2) Add the above-mentioned reagents R1 and R2 to the blood sample after treatment, and mix them in a reaction tube. Incubate at 37°C for 15 min. Then use chemiluminescence immunoassay to determine the luminescence value corresponding to the concentration of the standard solution. Calculate the valproic acid in the sample according to the standard curve.

[0058] Blood samples containing valproic acid at concentrations of 2.0 μg / mL to 150 μg / mL were prepared as samples. The samples were measured using the chemiluminescent immunoassay method with the kit of this embodiment. The results were compared with those measured using liquid chromatography-tandem mass spectrometry. The deviations between the concentration values ​​detected by the chemiluminescent immunoassay kit of this invention and the sample concentration values, the deviations between the concentration values ​​detected by the liquid chromatography-tandem mass spectrometry method of this invention and the sample concentration values, and the deviations between the concentrations detected by the chemiluminescent immunoassay kit of this invention and the liquid chromatography-tandem mass spectrometry method were calculated. The results are shown in Table 2.

[0059] Table 2

[0060]

[0061]

[0062] As shown in Table 2, the test kit of this embodiment can be used to automatically analyze and detect samples using the detection system of the chemiluminescence immunoassay instrument. The detected values ​​can stably fall within the acceptable error range of the true values, the test results are reliable, the detection efficiency is high, and it can reduce systematic errors and eliminate errors introduced by manual operation, thus showing broad application prospects.

[0063] Example 2

[0064] A valproic acid detection kit includes reagent R1 and reagent R2, wherein reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody, the concentration of the carboxyl magnetic bead is 1 mg / mL, and reagent R2 is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent, the protein concentration is 1 mg / mL.

[0065] The carboxyl magnetic bead reagent coated with valproic acid antibody, i.e., reagent R1, is prepared as follows:

[0066] (a1) Reagent preparation: Activation buffer: Prepare a 50mM MES buffer with pH 6 using MES and pure water as raw materials.

[0067] Buffer I: A 50 mM Tris buffer with pH 7 was prepared using Tris·HCl, Proclin 300, glucose, mannitol and EDTA as raw materials, with Proclin 300 0.01%, glucose 0.05%, mannitol 0.1% and EDTA 0.5%.

[0068] EDC solution: Prepare a 10 mg / mL EDC solution using EDC and activation buffer as raw materials;

[0069] NHS solution: A 10 mg / mL NHS solution was prepared using Sulfo-NHS and activation buffer as raw materials.

[0070] EDC and NHS mixture: Mix 10 mg / mL EDC solution with 10 mg / mL NHS solution at a volume ratio of 1:1;

[0071] (a2) Washing and activation of carboxyl magnetic beads: Take 10 mg of carboxyl magnetic beads with a particle size of 1 μm, add them to a 10 mL coated tube, magnetically separate them, and aspirate the supernatant. Then, take 2.5 mL of activation buffer and add it to the coated tube to resuspend the carboxyl magnetic beads. Mix at 2000 rpm for 10 s, magnetically separate them, and aspirate the supernatant. Take 100 μL of EDC and NHS mixture and add it to the coated tube. Mix at 2000 rpm for 10 s, and then place it on a roller at 25 °C for 30 min to activate. Take 2.5 mL of activation buffer and add it to the coated tube to resuspend it. Magnetically separate it, and aspirate the supernatant to obtain activated carboxyl magnetic beads.

[0072] (a3) Carboxyl magnetic beads coated with valproic acid antibody: The activated carboxyl magnetic beads were resuspended in 2.5 mL of activation buffer, 400 μg of valproic acid monoclonal antibody was added, and the mixture was mixed at 2000 rpm for 10 s. The mixture was then reacted on a roller at 20 ℃ for 3.5 h. After magnetic separation, the supernatant was collected, and the mixture was resuspended in 2.5 mL of buffer I. The mixture was then blocked on a roller at 20 ℃ for 2 h. After blocking, the mixture was magnetically separated, and the supernatant was collected to obtain carboxyl magnetic beads coated with valproic acid antibody.

[0073] (a4) Carboxyl magnetic bead reagent coated with valproic acid antibody: Take the above-mentioned carboxyl magnetic beads coated with valproic acid antibody, add buffer I to dilute them so that the concentration of carboxyl magnetic beads is 1 mg / mL, and obtain carboxyl magnetic bead reagent coated with valproic acid antibody.

[0074] The valproic acid-based antigen-alkaline phosphatase labeling reagent, i.e., reagent R2, is prepared as follows:

[0075] (b1) Reagent preparation: Buffer II: Prepare a 50mM Tris buffer with pH 7 using Tris·HCl, Proclin 300, mouse IgG, histidine, glycerol and PEG600 as raw materials, with Proclin 300 0.01%, mouse IgG 0.05μg / mL, histidine 0.05%, glycerol 10% and PEG600 0.1%;

[0076] (b2) Valproic acid synthesis antigen-alkaline phosphatase label: 100 μg of valproic acid synthesis antigen was pipetted into a 2 mL labeling tube, 0.5 mg of alkaline phosphatase was added, and then 1×TBS buffer was added to a total volume of 150 μL. After mixing, the mixture was placed in a dialysis bag, and dialysis was carried out overnight at 2–8 °C with stirring using 1 L of 1×TBS buffer. The dialyzed valproic acid synthesis antigen and alkaline phosphatase were then placed into a 2 mL labeling tube, 5 μg of glutaraldehyde was added, and the mixture was mixed for 10 s. The mixture was then placed in a 25 °C dark environment for 2 h. After dialysis, 1 L of 1×TBS buffer was added, and dialysis was carried out overnight at 2–8 °C with stirring. After dialysis, the valproic acid synthesis antigen-alkaline phosphatase label was obtained. The total protein content of the dialysis product (excluding the protein concentration of mouse IgG) was detected by UV spectrophotometer. The average value was taken from three measurements.

[0077] (b3) Valproic acid synthesis antigen-alkaline phosphatase labeling reagent: Based on the total protein content in the above valproic acid synthesis antigen-alkaline phosphatase labeling reagent, add buffer II to dilute it to a protein concentration of 1 mg / mL (excluding the protein concentration of mouse IgG) to obtain the valproic acid synthesis antigen-alkaline phosphatase labeling reagent.

[0078] The method for detecting valproic acid in serum using the above-mentioned valproic acid detection kit is as follows:

[0079] (1) Dissolve the standard containing valproic acid in pure water and prepare different concentrations as shown in Example 1 as standard solutions. Add the above-mentioned reagents R1 and R2 to the different standard solutions and place them in the reaction tube. Shake and mix well, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the standard solution concentration. Plot a standard curve of standard solution concentration and luminescence value.

[0080] (2) Add the above-mentioned reagents R1 and R2 to the treated blood to be tested into the reaction tube, shake to mix, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the concentration of the standard solution. Then calculate the valproic acid in the sample according to the standard curve.

[0081] Example 3

[0082] A valproic acid detection kit includes reagent R1 and reagent R2. Reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody, and the concentration of the carboxyl magnetic bead is 1 mg / mL. Reagent R2 is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent, and the protein concentration is 1 mg / mL.

[0083] The carboxyl magnetic bead reagent coated with valproic acid antibody, i.e., reagent R1, is prepared as follows:

[0084] (a1) Reagent preparation: Activation buffer: Prepare a 50mM MES buffer with pH 6 using MES and pure water as raw materials.

[0085] Buffer I: A 50 mM Tris buffer with pH 7 was prepared using Tris·HCl, Proclin 300, glucose, mannitol and EDTA as raw materials, with Proclin 300 0.05%, glucose 2.0%, mannitol 5% and EDTA 5%.

[0086] EDC solution: Prepare a 10 mg / mL EDC solution using EDC and activation buffer as raw materials;

[0087] NHS solution: A 10 mg / mL NHS solution was prepared using Sulfo-NHS and activation buffer as raw materials.

[0088] EDC and NHS mixture: Mix 10 mg / mL EDC solution with 10 mg / mL NHS solution at a volume ratio of 1:1;

[0089] (a2) Washing and activation of carboxyl magnetic beads: Take 10 mg of carboxyl magnetic beads with a particle size of 1 μm, add them to a 10 mL coated tube, magnetically separate them, and aspirate the supernatant. Then, take 2.5 mL of activation buffer and add it to the coated tube to resuspend the carboxyl magnetic beads. Mix at 2000 rpm for 10 s, magnetically separate them, and aspirate the supernatant. Take 40 μL of EDC and NHS mixture and add it to the coated tube. Mix at 2000 rpm for 10 s, and then place it on a roller at 25℃ for 30 min to activate. Take 2.5 mL of activation buffer and add it to the coated tube to resuspend it. Magnetically separate it, and aspirate the supernatant to obtain activated carboxyl magnetic beads.

[0090] (a3) Carboxyl magnetic beads coated with valproic acid antibody: The activated carboxyl magnetic beads were resuspended in 2.5 mL of activation buffer, 5 mg of valproic acid monoclonal antibody was added, and the mixture was mixed at 2000 rpm for 10 s. The mixture was then reacted on a roller at 30 ℃ for 3 h. After magnetic separation, the supernatant was aspirated, and the mixture was resuspended in 2.5 mL of buffer I. The mixture was then blocked on a roller at 30 ℃ for 2 h. After blocking, the mixture was magnetically separated, and the supernatant was aspirated to obtain carboxyl magnetic beads coated with valproic acid antibody.

[0091] (a4) Carboxyl magnetic bead reagent coated with valproic acid antibody: Take the above-mentioned carboxyl magnetic beads coated with valproic acid antibody, add buffer I to dilute them so that the concentration of carboxyl magnetic beads is 1 mg / mL, and obtain carboxyl magnetic bead reagent coated with valproic acid antibody.

[0092] The valproic acid-based antigen-alkaline phosphatase labeling reagent, i.e., reagent R2, is prepared as follows:

[0093] (b1) Reagent preparation: Buffer II: Prepare a 50mM Tris buffer at pH 7 using Tris·HCl, Proclin 300, mouse IgG, histidine, glycerol and PEG600 as raw materials, with Proclin 300 0.05%, mouse IgG 5μg / mL, histidine 5%, glycerol 50% and PEG600 5%;

[0094] (b2) Valproic acid synthetic antigen-alkaline phosphatase label: 100 μg of valproic acid synthetic antigen was pipetted into a 2 mL labeling tube, 0.1 mg of alkaline phosphatase was added, and then 1×TBS buffer was added to a total volume of 150 μL. After mixing, the mixture was placed in a dialysis bag, and dialysis was performed overnight at 2–8 °C with stirring using 1 L of 1×TBS buffer. The dialyzed valproic acid synthetic antigen and alkaline phosphatase were then placed into a 2 mL labeling tube, 1 μg of glutaraldehyde was added, and the mixture was mixed for 10 s. The mixture was then placed in a 25 °C dark environment for 2.5 h. After dialysis, 1 L of 1×TBS buffer was added, and dialysis was performed overnight at 2–8 °C with stirring. After dialysis, the valproic acid synthetic antigen-alkaline phosphatase label was obtained. The total protein content of the dialysis product (excluding the protein concentration of mouse IgG) was detected by UV spectrophotometer. The average value was taken from three measurements.

[0095] (b3) Valproic acid synthesis antigen-alkaline phosphatase labeling reagent: Based on the total protein content in the above valproic acid synthesis antigen-alkaline phosphatase labeling reagent, add buffer II to dilute it to a protein concentration of 1 mg / mL (excluding the protein concentration of mouse IgG) to obtain the valproic acid synthesis antigen-alkaline phosphatase labeling reagent.

[0096] The method for detecting valproic acid in serum using the above-mentioned valproic acid detection kit is as follows:

[0097] (1) Dissolve the standard containing valproic acid in pure water and prepare different concentrations as standard solutions in Example 1. Add the above-mentioned reagents R1 and R2 to the different standard solutions and place them in the reaction tube. Shake and mix well, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the standard solution concentration. Plot a standard curve of standard solution concentration and luminescence value.

[0098] (2) Add the above-mentioned reagents R1 and R2 to the treated blood to be tested into the reaction tube, shake to mix, incubate at 37°C for 15 min, and then use chemiluminescence immunoassay to test and determine the luminescence value corresponding to the concentration of the standard solution. Then calculate the valproic acid in the sample according to the standard curve.

[0099] Comparative Example 1

[0100] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, mannitol and EDTA as raw materials, and Proclin 300 0.1%, mannitol 2% and EDTA 1%; the rest are exactly the same.

[0101] Comparative Example 2

[0102] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, glucose and EDTA as raw materials, and Proclin 300 0.1%, glucose 1% and EDTA 1%; the rest are exactly the same.

[0103] Comparative Example 3

[0104] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, glucose and mannitol as raw materials, and Proclin 300 0.1%, glucose 1% and mannitol 2%; the rest are exactly the same.

[0105] Comparative Example 4

[0106] A valproic acid detection kit differs from Example 2 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, glucose, mannitol and EDTA as raw materials, and Proclin 300 0.01%, glucose 0.02%, mannitol 0.05% and EDTA 0.2%; the rest are exactly the same.

[0107] Comparative Example 5

[0108] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, histidine, glycerol and PEG600 as raw materials, and Proclin 300 0.1%, histidine 1%, glycerol 30% and PEG600 2%; the rest are exactly the same.

[0109] Comparative Example 6

[0110] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, mouse IgG, glycerol and PEG600 as raw materials, and Proclin 300 0.1%, mouse IgG 1μg / mL, glycerol 30% and PEG600 2%; the rest are exactly the same.

[0111] Comparative Example 7

[0112] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, mouse IgG, histidine, and PEG600 as raw materials, and Proclin 300 0.1%, mouse IgG 1μg / mL, histidine 1%, and PEG600 2%; the rest are exactly the same.

[0113] Comparative Example 8

[0114] A valproic acid detection kit differs from Example 1 in that: Buffer I: a 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, mouse IgG, histidine, and glycerol as raw materials, and Proclin 300 0.1%, mouse IgG 1μg / mL, histidine 1%, and glycerol 30%; the rest are exactly the same.

[0115] Comparative Example 9

[0116] A valproic acid detection kit differs from Example 2 in that: Buffer I: A 50mM Tris buffer with pH 7 is prepared using Tris·HCl, Proclin 300, mouse IgG, histidine, glycerol, and PEG600 as raw materials, wherein Proclin 300 0.01%, mouse IgG 0.02μg / mL, histidine 0.02%, glycerol 8%, and PEG600 0.05%.

[0117] 1. Blood samples containing valproic acid at concentrations of 5 μg / mL, 82 μg / mL, and 145 μg / mL were prepared as samples. The samples were analyzed using chemiluminescent immunoassay with the kits of Examples 1-3 and Comparative Examples 1-9, as shown in Tables 3-5.

[0118] Table 3

[0119]

[0120] Table 4

[0121]

[0122]

[0123] Table 5

[0124]

[0125] 2. In addition, the kits of Examples 1-3 and Comparative Examples 1-9 were placed at 37°C for 0 days, 1 day, 3 days, and 7 days, and blood samples containing valproic acid at a concentration of 25 μg / mL were measured using chemiluminescent immunoassay. The results are shown in Table 6. The kits of Examples 1-3 and Comparative Examples 1-9 were placed at 4°C for 0 months, 4 months, 8 months, and 12 months, and blood samples containing valproic acid at a concentration of 102 μg / mL were measured using chemiluminescent immunoassay. The results are shown in Table 7.

[0126] Table 6

[0127]

[0128]

[0129] Table 7

[0130]

[0131] As shown in Tables 3-5, 6, and 7, compared with Comparative Examples 1-4, Examples 1-3 use Buffer I containing appropriate proportions of glucose, mannitol, and EDTA as the protective working solution for the carboxyl magnetic beads coated with valproic acid antibodies. The synergistic effect of glucose, mannitol, and EDTA effectively improves the stability of the carboxyl magnetic beads coated with valproic acid antibodies, solving the problem of poor stability, easy formation of large amounts of non-specific binding, and high background values ​​associated with valproic acid antibody carboxyl magnetic beads. This significantly improves the detection stability and accuracy of the valproic acid detection kit of the present invention, further expanding its application scope. Compared with the comparative examples... Compared to Examples 5-9, Examples 1-3 use Buffer II containing appropriate proportions of mouse IgG, histidine, glycerol, and PEG600 as the enzyme working solution for the valproic acid synthesis antigen-alkaline phosphatase label. The simultaneous synergistic effect of mouse IgG, histidine, glycerol, and PEG600 effectively ensures the activity of alkaline phosphatase while also eliminating non-specific binding in valproic acid detection and reducing protein binding in blood samples. This significantly improves the detection accuracy of the valproic acid detection kit of this invention. Consequently, the valproic acid detection kit of this invention exhibits good stability and reliable detection results when stored at 4°C for one year.

[0132] In summary, the valproic acid detection kit of the present invention uses a simple reagent composition, eliminates the need for expensive instruments, reduces costs, and provides a rapid, convenient, stable, and accurate detection method for valproic acid in samples.

[0133] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit and essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0134] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A valproic acid detection kit, characterized in that, It includes reagent R1 and reagent R2, wherein reagent R1 is a carboxyl magnetic bead reagent coated with valproic acid antibody and contains buffer I, wherein buffer I is a Tris buffer containing Proclin 300, glucose, mannitol and EDTA. R2 reagent is a valproic acid synthesis antigen-alkaline phosphatase labeling reagent and contains buffer II, which is a Tris buffer containing Proclin 300, mouse IgG, histidine, glycerol and PEG 600.

2. The valproic acid detection kit according to claim 1, characterized in that, The method for preparing the carboxyl magnetic bead reagent coated with valproic acid antibody includes: washing and activating carboxyl magnetic beads, mixing the activated carboxyl magnetic beads with valproic acid antibody, and reacting at 20-30°C for 3-4 hours to obtain carboxyl magnetic beads coated with valproic acid antibody, blocking, and mixing the carboxyl magnetic beads coated with valproic acid antibody with buffer I to obtain the carboxyl magnetic bead reagent coated with valproic acid antibody. The mass ratio of the carboxyl magnetic beads to the valproic acid antibody is 50 to 2:

1.

3. A valproic acid detection kit according to claim 1 or 2, characterized in that, In the carboxyl magnetic bead reagent coated with valproic acid antibody, the concentration of carboxyl magnetic beads is 0.1–1 mg / mL.

4. The valproic acid detection kit according to claim 3, characterized in that, The buffer I is a 50-500 mM Tris buffer containing 0.01-0.1% Proclin 300, 0.05-2.0% glucose, 0.1-5% mannitol and 0.5-5% EDTA, with a pH of 6-8.

5. The valproic acid detection kit according to claim 2, characterized in that, The cleaning and activation treatment of the carboxyl magnetic beads includes: taking the carboxyl magnetic beads and resuspending them in an activation buffer solution for cleaning, then adding a mixture of EDC and NHS to the cleaned carboxyl magnetic beads and activating them for 20-30 minutes; the mass ratio of the carboxyl magnetic beads to EDC is 10-50:1, and the mass ratio of EDC to NHS is 1:

1.

6. The valproic acid detection kit according to claim 1, characterized in that, The preparation method of the valproic acid synthesis antigen-alkaline phosphatase labeling reagent includes: mixing valproic acid synthesis antigen, alkaline phosphatase, and TBS buffer and dialyzing, then adding a coupling agent and reacting in the dark for 2-3 hours, and after the reaction, adding TBS buffer and dialyzing to obtain valproic acid synthesis antigen-alkaline phosphatase labeling, and mixing the valproic acid synthesis antigen-alkaline phosphatase labeling with buffer II to obtain the valproic acid synthesis antigen-alkaline phosphatase labeling reagent; The mass ratio of the valproic acid-synthesized antigen to alkaline phosphatase is 1:1 to 10.

7. A valproic acid detection kit according to claim 1 or 6, characterized in that, In the valproic acid synthesis antigen-alkaline phosphatase labeling reagent, the protein concentration is 0.1–2 mg / ml.

8. The valproic acid detection kit according to claim 7, characterized in that, The buffer II is a 50-500 mM Tris buffer containing 0.01-0.1% Proclin 300, 0.05-5 μg / mL mouse IgG, 0.05-5% histidine, 10-50% glycerol and 0.1-5% PEG 600, with a pH of 6-8.

9. The valproic acid detection kit according to claim 6, characterized in that, The coupling agent is 0.05-1% of the total mass of valproic acid synthetic antigen and alkaline phosphatase; the coupling agent is one of glutaraldehyde, sodium periodate or N-hydroxysuccinimide maleimide, preferably glutaraldehyde.

10. The application of the valproic acid detection kit according to claim 1, characterized in that, The valproic acid detection kit utilizes chemiluminescent immunoassay to detect valproic acid in serum.

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