Reagent R1 for detecting valproic acid by latex-enhanced immunoturbidimetry, kit and application thereof

Abstract

The present invention discloses a reagent R1, a kit and an application thereof for detecting valproic acid by latex enhanced immunoturbidimetry. The reagent R1 includes a dissociator of protein-bound valproic acid, and the dissociator includes 1) acetylsalicylic acid or 2) cimetidine and erythromycin. The kit of the present invention can more accurately detect the total valproic acid drug content in a free state in plasma or serum, and is closer to the HPLC-MS / MS measurement value than a commercial kit, and can be quickly applied to the detection of a conventional automatic biochemical analyzer, providing a more accurate reference value for clinical use on the basis of convenient detection.

Description

Technical Field

[0001] The invention relates to the technical field of valproic acid detection, in particular to a reagent R1 for detecting valproic acid by latex enhanced immunoturbidimetry, a kit containing the reagent R1 and applications thereof. Background Art

[0002] Valproic acid is a broad-spectrum antiepileptic drug that does not contain nitrogen. It is the first choice for primary grand mal seizures and absence seizures. Valproic acid is a short-chain fatty acid and is the only antiepileptic drug that does not contain aromatic rings or nitrogen elements. The efficacy of valproic acid treatment in patients depends on the type of epilepsy, the severity of the disease, and individual pharmacokinetic differences. In addition to anti-epileptic drugs, this drug can also be used to treat febrile convulsions, movement disorders, chorea, porphyria, schizophrenia, pain caused by herpes zoster, adrenal dysfunction, and prevent alcohol withdrawal syndrome. Studies have shown that the correlation between valproic acid dose and blood concentration is poor, but its efficacy and adverse reactions are highly correlated with blood concentration. Too low concentration will cause insufficient efficacy, and too high concentration is prone to toxic reactions. This result supports the necessity of regular monitoring of valproic acid blood concentration. Valproic acid is a slow-acting drug that may not be able to effectively control epileptic seizures until 6 months after reaching the appropriate drug concentration. Monitoring valproic acid in the blood can enable clinicians to promptly understand the individual differences in patients' drug dosages and adjust the drug dosage for different patients based on individual differences. This can effectively improve the treatment effect and is of great significance for rational clinical drug use.

[0003] Currently, the immunoassays used to determine the blood concentration of valproic acid mainly include chromatography (high performance liquid chromatography HPLC; gas chromatography GC; liquid chromatography-mass spectrometry analysis), homogeneous enzyme immunoassay (HEIA), chemiluminescence immunoassay (CLIA), and fluorescence polarization immunoassay (FPIA).

[0004] At present, valproic acid has no absorption under ultraviolet light because it does not have an aromatic ring or conjugated double bond structure. The general HPLC method cannot be directly monitored by an ultraviolet detector. Derivatization treatment is required before determination, which is relatively cumbersome and limits the use of HPLC in large clinical samples. Although liquid chromatography-mass spectrometry (LC-MS / MS) has the advantages of high sensitivity, strong specificity, fast analysis speed and high throughput, the instrument is expensive and has a low domestic penetration rate. Sample analysis using chromatographic analysis requires sample pretreatment, and the drug content in the sample finally detected includes free and protein-bound states, which is the total drug content in the sample.

[0005] For existing immunoassay methods, chemiluminescence and homogeneous enzyme immunoassay are fast in analysis speed and highly automated compared to chromatography, but the kits are expensive and require the use of specially matched instruments and equipment, and the valproic acid that can be free in the sample cannot be accurately detected. The homogeneous enzyme immunoassay sample also needs to be diluted and measured, so that the detection cannot quickly and accurately draw conclusions. Moreover, what is measured for biochemical analysis and immunoassay techniques (homogeneous enzyme immunoassay (HEIA), chemiluminescence immunoassay (CLIA), fluorescence polarization immunoassay (FPIA), etc.) is the valproic acid drug content in the free state in the sample, which is not accurate enough. Similarly, the latex enhanced immune turbidimetric kit for commercial detection of valproic acid on the market can only detect the valproic acid drug concentration of the free part in the sample, which is not good in accuracy, and the kit is expensive, and requires the use of specially matched instruments and equipment, which is not conducive to promotion and use. Summary of the invention

[0006] In view of the shortcomings of the prior art, a reagent R1 for detecting valproic acid by latex-enhanced immunoturbidimetry for detecting valproic acid blood concentration in a conventional automatic biochemical analyzer, a kit containing the reagent R1 and the application thereof are provided. The reagent R1 can more accurately detect the total valproic acid drug content in a free state in plasma or serum, is simple to operate, has higher accuracy, and can be quickly applied to a conventional automatic biochemical analyzer.

[0007] After the valproic acid drug enters the macrocirculation through the administration site, a part of it exerts its efficacy or toxicity by binding to receptors or reaching the affected area in a free state; a part of it temporarily does not exert its efficacy or toxicity by binding to plasma proteins; there is a dynamic balance between the free state of drugs, drugs bound to plasma proteins, metabolized drugs and excretion, and it varies with individual constitutions. Therefore, the valproic acid measured by most existing immunoassay methods is the free state of valproic acid concentration in a single time point sample, and cannot reflect the free state of valproic acid concentration at a dynamic point, resulting in the inability to correctly judge all free-state valproic acid concentrations in the blood that can produce efficacy or toxicity, which affects clinical medication. Therefore, the inventors try to use a reagent / method with strong anti-binding protein interference ability, which can dissociate the valproic acid drug in a state bound to plasma proteins, so as to accurately detect the total free state of valproic acid drug content in plasma or serum.

[0008] In order to solve the above defects, the first aspect of the present invention provides a reagent R1 for detecting valproic acid by latex enhanced immunoturbidimetry, which includes a dissociator of protein-bound valproic acid, wherein the dissociator includes 1) acetylsalicylic acid, or 2) cimetidine and erythromycin.

[0009] Preferably, the sample to be tested is from blood, such as plasma or serum.

[0010] When the dissociating agent is acetylsalicylic acid, the content of acetylsalicylic acid in the reagent R1 is preferably 1-5 mg / mL, more preferably 1-2 mg / mL, and even more preferably 2 mg / mL.

[0011] When the dissociating agents are cimetidine and erythromycin, the contents in the reagent R1 are: preferably 0.1-1 mg / mL of cimetidine, and 0.1-0.5 mg / mL of erythromycin; more preferably, 0.2-0.4 mg / mL of cimetidine, and 0.1-0.2 mg / mL of erythromycin; and even more preferably, 0.35 mg / mL of cimetidine, and 0.2 mg / mL of erythromycin.

[0012] The reagent R1 of the present invention may also include substances conventionally used in existing latex-enhanced immunoturbidimetric detection kits, especially latex-enhanced immunoturbidimetric reagent R1 for detecting valproic acid, such as:

[0013] (1) a stabilizer, used to stabilize the detection system, especially to protect the protein and maintain the protein structure. The concentration of the stabilizer is usually 1-10%. The stabilizer is preferably chloride and / or disaccharide. The chloride can be selected from one or more of sodium chloride, potassium chloride and magnesium chloride, more preferably sodium chloride or potassium chloride, and even more preferably sodium chloride. The disaccharide is preferably selected from one or more of sucrose, maltose and trehalose, more preferably sucrose.

[0014] (2) a buffer for preparing a buffer system of reagent R1. The concentration of the buffer is usually 0.2-1%. The buffer is preferably one or more selected from MES, Tris, MOPS and HEPES, more preferably MES, and even more preferably MES monohydrate;

[0015] (3) a surfactant, wherein the concentration of the surfactant is usually 0.01-0.1%, and the surfactant is preferably a nonionic surfactant, such as a polyoxyethylene nonionic surfactant, a polyol nonionic surfactant, or an alkylolamide nonionic surfactant, more preferably a polyol nonionic surfactant, and even more preferably Tween, such as Tween 20;

[0016] (4) a metal ion chelator, wherein the concentration of the metal ion chelator is generally 0.1-1%, and the metal ion chelator is preferably EDTA or its potassium salt, and the potassium salt thereof is more preferably EDTA-2K or EDTA-3K, and even more preferably EDTA-2K; and / or,

[0017] (5) a coagulant, wherein the concentration of the coagulant is 1-5%, and the coagulant is, for example, a polysaccharide or polyvinyl pyrrolidone. Preferably, the polysaccharide is dextran, more preferably dextran with a molecular weight of 450,000-650,000;

[0018] Furthermore, reagent R1 may also include:

[0019] (6) Valproic acid antigen (as a detection antigen), the concentration is usually 130-150 ng / mL;

[0020] (7) a preservative, wherein the concentration of the preservative is generally 0.01-0.1%, and the preservative is preferably one or more selected from gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3(2H)-isothiazolone 5-bromo-5-nitro-1,3-dioxane and Proclin-300, and more preferably sodium azide; and / or

[0021] (8) A blocking agent, the concentration of which is usually 0.01-0.1%, such as BSA or ethanolamine, preferably ethanolamine.

[0022] The above percentages are the mass volume percentages in the reagent R1, in g / mL. The above stabilizers, buffers, surfactants, metal ion chelators, coagulants, preservatives, sealants and other components can be replaced with conventional substances and conventional concentrations in the reagent R1 of the existing latex-enhanced immunoturbidimetric method for detecting valproic acid. In the detection kit, only the buffer can be included, and then configured into a buffer during the detection; it can also be replaced with a pre-configured buffer.

[0023] In a preferred embodiment,

[0024] The sodium chloride is 1-5%, the sucrose is 1-5%, the dextran is 1-5%, the MES monohydrate is 0.2-1%, the Tween 20 is 0.01-0.1%, the EDTA-2K is 0.1-1%, the sodium azide is 0.01-0.1%, the ethanolamine is 0.01-0.1%, the valproic acid antigen is 130-150 ng / mL, and the pH of the reagent R1 is 5-9.

[0025] Preferably, the reagent R1 comprises:

[0026] 1-2 mg / mL acetylsalicylic acid, 1-3% sodium chloride, 1-3% sucrose, 2.5-3% dextran, 0.3-0.6% MES monohydrate, 0.02-0.05% Tween 20, 0.3-0.5% EDTA-2K, 0.03-0.05% sodium azide, 0.01-0.05% ethanolamine, and 140-145 ng / mL valproic acid antigen, pH 6-7; or,

[0027] 0.2-0.4 mg / mL cimetidine, 0.1-0.2 mg / mL erythromycin, 1-3% sodium chloride, 1-3% sucrose, 2.5-3% dextran, 0.3-0.6% MES monohydrate, 0.02-0.05% Tween 20, 0.3-0.5% EDTA-2K, 0.03-0.05% sodium azide, 0.01-0.05% ethanolamine, and 140-145 ng / mL valproic acid antigen, pH 6-7.

[0028] Most preferably, the reagent R1 comprises:

[0029] 2 mg / mL acetylsalicylic acid, 1% sodium chloride, 1% sucrose, 2.5% dextran, 0.533% MES monohydrate, 0.02% Tween 20, 0.5% EDTA-2K, 0.05% sodium azide, 0.05% ethanolamine, and 145 ng / mL valproic acid antigen, pH 6.8; or,

[0030] 0.35 mg / mL cimetidine, 0.2 mg / mL erythromycin, 1% sodium chloride, 1% sucrose, 2.5% dextran, 0.533% MES monohydrate, 0.02% Tween 20, 0.5% EDTA-2K, 0.05% sodium azide, 0.05% ethanolamine, and 145 ng / mL valproic acid antigen, pH 6.8.

[0031] A second aspect of the present invention provides a valproic acid detection kit, which includes the reagent R1 described in the present invention.

[0032] Preferably, the reagent R2 in the existing latex enhanced immunoturbidimetry kit for detecting valproic acid is also included.

[0033] The reagent R2 generally comprises latex microspheres, a protein stabilizer, a buffer, a surfactant, a metal ion chelator and / or a preservative.

[0034] Among them, the dosage of the latex microspheres is usually 5-20 μL / mL, the concentration of the protein stabilizer is usually 1-5%, the concentration of the buffer is usually 0.2-1%, the concentration of the surfactant is usually 0.001-0.01%, the concentration of the metal ion chelator is usually 0.1-0.5%, and the concentration of the preservative is usually 0.01-0.1%.

[0035] The above percentages are the mass volume percentages in reagent R2, in g / mL. The above protein stabilizers, buffers, surfactants, metal ion chelators, preservatives and other components can be replaced with conventional substances and conventional concentrations in the reagent R2 of the existing latex enhanced immunoturbidimetric method for detecting valproic acid.

[0036] Preferably, the pH of the reagent R2 is 4-6, and the reagent R2 contains:

[0037] The latex microspheres used in the turbidimetric method generally refer to sensitized latex microspheres, that is, latex microspheres coupled with antibodies or antigens. In the present invention, the latex microspheres are coupled with valproic acid or its antibodies, which are called valproic acid sensitized latex microspheres;

[0038] The protein stabilizer is betaine hydrochloride, preferably betaine hydrochloride;

[0039] The buffer is one or more of MES, Tris, MOPS, and HEPES, for example, HEPES;

[0040] The surfactant is a nonionic surfactant such as a polyoxyethylene nonionic surfactant, a polyol nonionic surfactant or an alkylolamide nonionic surfactant, preferably a polyol nonionic surfactant, more preferably Tween such as Tween 20;

[0041] The metal ion chelator is, for example, EDTA or its potassium salt. Preferably, the potassium salt is EDTA-2K or EDTA-3K, for example, EDTA-2K.

[0042] The preservative is selected from one or more of gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3(2H)-isothiazolone, 5-bromo-5-nitro-1,3-dioxane and Proclin-300, for example, sodium azide;

[0043] More preferably,

[0044] The pH of the reagent R2 is 5-5.5,

[0045] The dosage of the valproic acid sensitized latex microspheres is preferably 5-10 μL / mL, the betaine hydrochloride is 1-5%, the HEPES is 0.2-1%, the Tween 20 is 0.001-0.01%, the EDTA-2K is 0.1-0.5%, the sodium azide is 0.01-0.1%, and the pH is 7-9.

[0046] In a preferred embodiment of the present invention, the pH of the reagent R2 is 5.5, the dosage of the valproic acid sensitized latex microspheres is 10 μL / mL, the betaine hydrochloride is 2%, the HEPES is 0.596%, the Tween 20 is 0.005%, the EDTA-2K is 0.3%, the sodium azide is 0.05%, and the pH is 8.

[0047] The above percentage is the mass volume percentage in reagent R2, in g / mL. The amount of the above valproic acid sensitized latex microspheres is based on the raw material of latex microspheres, that is, the ratio of the volume of the raw material latex microspheres to the volume of the buffer system in the R2 reagent.

[0048] In a specific embodiment, the preparation method of the valproic acid sensitized latex microspheres comprises:

[0049] (1) mixing buffer B and the latex microspheres, adding EDC and NHS and shaking for 20-50 minutes, preferably 35 minutes, adding surfactant A, reacting for 10-30 minutes, preferably 20 minutes, and sonicating 10 times;

[0050] (2) adding valproic acid monoclonal antibody to carry out cross-linking reaction;

[0051] Preferably, it further comprises adding a blocking agent to the reagent R2 for blocking;

[0052] The buffer B is NaHCO 3 solution, the solid content of the latex microspheres is 5%, the surfactant A is poloxamer 188, the dosage ratio of EDC to latex microspheres is 0.5 μg / μL, the weight ratio of EDC to NHS is 1:10, the dosage ratio of poloxamer 188 to latex microspheres is 100-500 μg / μL, and the dosage ratio of valproic acid monoclonal antibody to latex microspheres is 0.5-2.5 μg / μL;

[0053] The cross-linking reaction was performed by shaking for 20 minutes, ultrasonicating for 10 times, shaking for 60 minutes, ultrasonicating for 10 times, and finally shaking for 30 minutes.

[0054] The latex microspheres may be made of materials and have particle sizes commonly used in the art.

[0055] The third aspect of the present invention provides a reagent for detecting valproic acid, which comprises a dissociating agent for protein-bound valproic acid and a reagent A, wherein the reagent A is a valproic acid standard and / or a valproic acid antibody, and the dissociating agent comprises 1) acetylsalicylic acid, or 2) cimetidine and erythromycin.

[0056] Preferably, the detection method is latex enhanced immunoturbidimetry; and / or, the detected sample is from blood such as plasma or serum.

[0057] The fourth aspect of the present invention provides the use of (1) acetylsalicylic acid or (2) cimetidine and erythromycin in the detection of valproic acid.

[0058] Preferably, the sample to be detected is from blood, such as plasma or serum; and / or, the detection method is latex enhanced immunoturbidimetry.

[0059] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0060] The reagents and raw materials used in the present invention are commercially available.

[0061] The positive and progressive effects of the present invention are:

[0062] The kit prepared by using the reagent R1 of the present invention contains a drug that competes with the protein for binding, so that the valproic acid drug in the state of being bound to the plasma protein can be dissociated, and the total valproic acid drug content in the free state in the plasma or serum can be detected, and the sample correlation with the HPLC-MS / MS measurement value is good, and the measurement value is relatively close. The operation is simple, and it can be quickly applied to the detection of a conventional automatic biochemical analyzer, and can provide a more accurate reference value for clinical use on the basis of convenient detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 A is a standard product of the detection kit (R1 contains 2 mg / mL acetylsalicylic acid) in the embodiment of the present invention for multi-point calibration, and a spline function is used for calculation to obtain a calibration curve schematic diagram;

[0064] Figure 1 B is a standard product of the detection kit (R1 contains 1 mg / mL acetylsalicylic acid) in the embodiment of the present invention for multi-point calibration, and is calculated using a spline function to obtain a calibration curve schematic diagram;

[0065] Figure 1 C is a standard product containing 0.35 mg / mL cimetidine and 0.2 mg / mL erythromycin in the detection kit (R1 in the embodiment of the present invention, and multi-point calibration is performed, and the spline function is used for calculation to obtain a calibration curve schematic diagram;

[0066] Figure 1 D is a standard product containing 0.2 mg / mL cimetidine and 0.1 mg / mL erythromycin in the detection kit (R1 in the embodiment of the present invention, and multi-point calibration is performed, and the spline function is used for calculation to obtain a calibration curve schematic diagram;

[0067] Figure 1E is a standard product containing 0.4 mg / mL cimetidine and 0.2 mg / mL erythromycin in the detection kit (R1 in the embodiment of the present invention, and multi-point calibration is performed, and the spline function is used for calculation to obtain a calibration curve schematic diagram.

[0068] Figure 2 A is a dot plot of comparison results between the measured value of the kit of the present invention (R1 does not contain acetylsalicylic acid or cimetidine and erythromycin) and the measured value of HPLC-MS / MS in the embodiment of the present invention;

[0069] Figure 2 B is a dot plot of the comparison results between the measured value of the kit of the present invention (R1 contains 2 mg / mL acetylsalicylic acid) and the measured value of HPLC-MS / MS in the embodiment of the present invention;

[0070] Figure 2 C is a dot plot of the comparison results between the measured value of the kit of the present invention (R1 contains 0.35 mg / mL cimetidine and 0.2 mg / mL erythromycin) and the measured value of HPLC-MS / MS in the embodiment of the present invention;

[0071] Figure 2 D is a dot plot of the comparison results between the measured values ​​of the commercial kit in the embodiment of the present invention and the measured values ​​of HPLC-MS / MS. DETAILED DESCRIPTION

[0072] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0073] 1. Material Preparation

[0074] 1.1 Preparation of valproic acid antigen

[0075] Preparation method of valproic acid antigen (see Wu FB, Yang YY, Wang XB, et al. A sample processing method for immunoassay of whole blood tacrolimus [J]. Analytical Biochemistry, 2019, 576: 13-19.):

[0076] (A) Accurately weigh 20 mg of valproic acid standard material (purchased from the National Pharmaceutical Standard Material Network: sodium valproate 100963-202004-50 ml) and 8 mg of succinic anhydride, add 0.7 ml of toluene and 1 ml of CH 2 Cl 25 mg of dimethylaminopyridine and 4 μl of triethylamine were added, and the mixture solution was stirred at room temperature under argon for 24 hours.

[0077] (B) The crude product was washed twice with 0.1 mol HCl, and the light yellow upper organic layer was collected and MgCl2 was added. 2 SO 4 Drying and evaporation to dryness by a stream of argon gave a pale yellow solid.

[0078] (C) 4.5 mg of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and 15 mg of N-hydroxysuccinimide were added to 0.5 ml of anhydrous dimethylformamide containing 5 mg of the pale yellow solid prepared as described above.

[0079] (D) After 30 min of reaction, the mixed solution was added dropwise to 4 ml NaHCO containing 20 mg KLH. 3 The mixture was incubated in a 0.1 M solution (pH 8.65) at room temperature for 90 min to obtain the valproic acid-KLH conjugate.

[0080] (E) The prepared valproic acid-KLH conjugate was purified by dialysis. Specifically, the prepared valproic acid-KLH conjugate was purified by gel filtration on a Sepharose CL-6B column (Shanghai Huamei Laboratory Instrument Factory Sepharose CL-6B column, 2.5×55 cm), and elution was monitored using AKTA-prime Plus (elution buffer: 50 mM Tris-HCl, pH 8.0, containing 0.9% NaCl and 0.05% NaN3, to obtain valproic acid antigen.

[0081] 1.2 Preparation method of valproic acid monoclonal antibody

[0082] Preparation method of valproic acid monoclonal antibody (valproic acid-McAb) (see Wu FB, Yang YY, Wang XB, et al. A sample processing method for immunoassay of whole blood tacrolimus [J]. Analytical Biochemistry, 2019, 576: 13-19. 2.2 Preparation of anti-FK506McAb):

[0083] The valproic acid antigen obtained in the preparation of 1.1 (E) of the above materials is used as an immunogen, and immunization and detection are performed by micro-long-range immunization, followed by cell fusion and screening, and monoclonal cell line ascites production and purification.

[0084] The valproic acid-McAb used in this example was developed on Balb / c mice. 50 μg of the above immunogen was prepared in an emulsion of 100 μl Freund's complete adjuvant for the first subcutaneous immunization, and 30 μg of the immunogen was prepared in an emulsion of 100 μl incomplete Freund's adjuvant for booster immunization every 4 weeks. A total of 4 immunizations were performed, and one week before cell fusion, 30 μg of the immunogen in 100 μl Hanks' balanced salt solution (Beyotime Biotechnology, China) was subcutaneously injected for the final booster.

[0085] The spleen cells of the immunized mice were electrofused with myeloma cells, and the fusion was screened by ELISA method to obtain the target monoclonal cell line with successful fusion. The target cell line was expanded and cultured, and after reaching a certain number, it was injected into the peritoneal cavity of the mouse for antibody enrichment. After taking out the ascites (enriched antibodies), the monoclonal antibody was purified by the caprylic acid-ammonium sulfate method to obtain the valproic acid monoclonal antibody coupled with R2 latex coating used in the kit.

[0086] Of course, the above-mentioned valproic acid antigen and valproic acid monoclonal antibody may also be commercially available products.

[0087] Example 1

[0088] The latex enhanced immune turbidimetric kit for determining valproic acid prepared by the invention comprises a reagent R1 (containing a dissociation component), R2 (antibody latex reagent) and a valproic acid calibrator.

[0089] 1. Preparation of Reagent R1

[0090] The components of R1 in Table 1 below were fully mixed with water, wherein the content percentages are the mass volume percentages in reagent R1, in units of g / mL.

[0091] Table 1 Main ingredients of reagent R1 (including dissociating agent acetylsalicylic acid) (pH 6.8)

[0092] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Acetylsalicylic acid 2mg / mL Valproic acid antigen 145 ng / mL

[0093] 2. Preparation of Reagent R2

[0094] Table 2 Main components of reagent R2 (antibody latex reagent) (pH 5.5)

[0095]

[0096]

[0097] The content percentage is the mass volume percentage in reagent R2, and the unit is g / mL.

[0098] Preparation process of valproic acid-sensitized latex microspheres (latex-coated coupled antibodies) in reagent R2:

[0099] A. Add 800 μL of NaHCO to the centrifuge tube. 3 The buffer solution (pH 8.0-9.0, concentration 20 mM) and 200 μL of latex microspheres (Jsrlifesciences, model: J1910N-08P0112, solid content 5%) were mixed, and 0.1 mg of EDC and 1 mg of NHS were added, and the mixture was shaken and reacted at room temperature for 35 min;

[0100] B. Add 20-100 mg of poloxamer 188, react for 20 min, and sonicate 10 times;

[0101] C. Cross-linking: Add 0.1-0.5 mg of the valproic acid monoclonal antibody prepared in 1.2 to the above materials and mix immediately, shake at room temperature for 20 minutes, sonicate 10 times, shake at room temperature for 60 minutes, sonicate 10 times again, and finally shake at room temperature for 30 minutes;

[0102] D. Blocking: 1 mL of the cross-linked latex reagent obtained in step C above was added with 60-120 μL of the blocking agent, mixed, sonicated 10 times, and reacted at room temperature for 60 min; centrifuged twice, ultracentrifuged for 40 min, and precipitated into valproic acid-sensitized latex microspheres (latex-coated coupled antibodies);

[0103] The blocking agent is a 0.01 mol / L boric acid buffer solution containing 1% BSA and 66 mM ethanolamine, with a pH of 8.0, which plays a quenching and blocking role.

[0104] Preparation process of reagent R2 (antibody latex reagent):

[0105] Add an appropriate amount of 20 mM HEPES to the valproic acid-sensitized latex microspheres prepared above until they can be blown apart, then ultracentrifuge, discard the supernatant, and precipitate with 20 ml of R2 buffer system (containing 0.596% HEPES, 0.05% NaN 3 , 0.005% T-20, 0.3% EDTA-2K, 2% betaine hydrochloride aqueous solution) and stored at 4°C.

[0106] Example 2

[0107] 2.1 Preparation of Reagent R1

[0108] Each component of R1 in Table 3 below was fully mixed with water. During the test, 145 ng / mL of the test antigen (valproic acid antigen) was added to the reagent R1.

[0109] Table 3 Composition of reagent R1 (containing dissociating agent acetylsalicylic acid) (pH 6.8)

[0110]

[0111]

[0112] 2.2 Preparation of reagent R2

[0113] The formula and preparation are the same as in Example 1.2.

[0114] Example 3

[0115] 3.1 Preparation of Reagent R1

[0116] The components of R1 in Table 4 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0117] Table 4 Main ingredients of reagent R1 (including dissociating agent acetylsalicylic acid) (pH 6.8)

[0118] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Acetylsalicylic acid 1.0mg / mL

[0119] 3.2 Preparation of reagent R2

[0120] The formula and preparation are the same as in Example 1.2.

[0121] Example 4

[0122] 4.1 Preparation of Reagent R1

[0123] The components of R1 in Table 5 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0124] Table 5 Main ingredients of reagent R1 (including dissociating agent acetylsalicylic acid) (pH 6.8)

[0125]

[0126]

[0127] 4.2 Preparation of Reagent R2

[0128] The formula and preparation are the same as in Example 1.2.

[0129] Example 5

[0130] 5.1 Preparation of Reagent R1

[0131] The components of R1 in Table 6 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0132] Table 6 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0133] Component name content MES 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Cimetidine 0.35mg / mL Erythromycin 0.2mg / mL

[0134] 5.2 Preparation of Reagent R2

[0135] The formula and preparation are the same as in Example 1.2.

[0136] Example 6

[0137] 6.1 Preparation of Reagent R1

[0138] The components of R1 in Table 7 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0139] Table 7 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0140]

[0141]

[0142] 6.2 Preparation of Reagent R2

[0143] The formula and preparation are the same as in Example 1.2.

[0144] Example 7

[0145] 7.1 Preparation of Reagent R1

[0146] The components of R1 in Table 8 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0147] Table 8 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0148] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Cimetidine 0.15mg / mL Erythromycin 0.1mg / mL

[0149] 7.2 Preparation of Reagent R2

[0150] The formula and preparation are the same as in Example 1.2.

[0151] Example 8

[0152] 8.1 Preparation of Reagent R1

[0153] The components of R1 in Table 9 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0154] Table 9 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0155]

[0156]

[0157] 8.2 Preparation of Reagent R2

[0158] The formula and preparation are the same as in Example 1.2.

[0159] Example 9

[0160] 9.1 Preparation of Reagent R1

[0161] The components of R1 in Table 10 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0162] Table 10 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0163] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Cimetidine 0.2mg / mL Erythromycin 0.1mg / mL

[0164] 9.2 Preparation of Reagent R2

[0165] The formula and preparation are the same as in Example 1.2.

[0166] Example 10

[0167] 10.1 Preparation of Reagent R1

[0168] The components of R1 in Table 11 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0169] Table 11 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0170]

[0171]

[0172] 10.2 Preparation of Reagent R2

[0173] The formula and preparation are the same as in Example 1.2.

[0174] Embodiment 11

[0175] 11.1 Preparation of Reagent R1

[0176] The components of R1 in Table 12 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0177] Table 12 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0178] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Cimetidine 0.5mg / mL Erythromycin 0.2mg / mL

[0179] 11.2 Preparation of Reagent R2

[0180] The formula and preparation are the same as in Example 1.2.

[0181] Example 12

[0182] 12.1 Preparation of Reagent R1

[0183] The components of R1 in Table 13 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0184] Table 13 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0185]

[0186]

[0187] 12.2 Preparation of Reagent R2

[0188] The formula and preparation are the same as in Example 1.2.

[0189] Embodiment 13

[0190] 13.1 Preparation of Reagent R1

[0191] The components of R1 in Table 14 below were mixed with water. During the test, 145 ng / mL of the test antigen was added to the reagent R1.

[0192] Table 14 Main ingredients of reagent R1 (including dissociating agents cimetidine and erythromycin) (pH 6.8)

[0193] Component name content MES monohydrate 0.533％ NaCl 1.0％ Ethanolamine 0.05％ <![CDATA[NaN 3 ]]> 0.05％ T-20 0.02％ EDTA-2K 0.5％ sucrose 1％ Dextran 2.5％ Cimetidine 0.4mg / mL Erythromycin 0.2mg / mL

[0194] 13.2 Preparation of Reagent R2

[0195] The formula and preparation are the same as in Example 1.2.

[0196] Application Example 1 Preparation of Calibration Curve and Standard Addition Recovery Determination

[0197] Proposed calibration curve results:

[0198] The reagent prepared in the above embodiment was tested with a Hitachi 7180 fully automatic biochemical analyzer, the test wavelength was 546nm, the sample volume or calibration product was 9μL, and then 180μL of R1 reagent was added, and the mixture was incubated at 37°C for 5min, and then 80μL of R2 reagent was added. The absorbance A1 value was read after 20S, and the absorbance A2 was read after 4 minutes and 42 seconds of incubation at 37°C. Then, the absorbance ΔA=A2-A1;

[0199] Use the standard to perform multi-point calibration and obtain the OD value of the calibration product, as shown in Table 15 below. Use the spline function to calculate and obtain the calibration curve, as shown in Figure 1 A (Example 1), Figure 1 B (Example 3) and Figure 1 C (Example 5), Figure 1 D (Example 9), Figure 1 E (Example 13).

[0200] Table 15 OD values ​​of calibrants obtained by multi-point calibration using standard products

[0201]

[0202]

[0203] From the results in Table 15, the inhibition rates (F / A) of Examples 1, 3, 5, 9 and 13 are ≤20%, and the difference between point A and point B is moderate (between 0.85≤B / A≥0.65), which shows a good fit of the calibration curve.

[0204] Spike recovery determination:

[0205] Pure valproic acid (National Drug Standard Material Network, sodium valproate 100963-202004) was added to the blank sample to make the final concentration of valproic acid in plasma 12.5μg / mL, 50μg / mL and 150μg / mL to detect the recovery rate and coefficient of variation of valproic acid. The recovery rate and coefficient of variation of each concentration were calculated based on 5 repeated data of 3 different days, and the recovery rate was quantitatively calculated according to the linear equation of the drawn working curve. The results are as follows:

[0206] Table 16 Example 1 Valproic acid test value - R1 contains 2 mg / mL acetylsalicylic acid

[0207]

[0208]

[0209] Table 17 Example 3 Valproic acid test value - R1 contains 1 mg / mL acetylsalicylic acid

[0210]

[0211] Table 18 Example 5 Valproic acid test values ​​- R1 contains cimetidine (0.35 mg / mL) and erythromycin (0.2 mg / mL)

[0212]

[0213] Table 19 Example 9 Valproic acid test values ​​- R1 contains cimetidine (0.2 mg / mL) and erythromycin (0.1 mg / mL)

[0214]

[0215] Table 20 Example 13 Valproic acid test values ​​- R1 contains cimetidine (0.4 mg / mL) and erythromycin (0.2 mg / mL)

[0216]

[0217]

[0218] From the measurement results in Tables 16 to 20 above, the coefficient of variation of the kits in the above embodiments is lower than 10%, and the recovery rate is 115%≤≥85%, which shows good repeatability and accuracy; among them, the recovery rates of Embodiment 1 and Embodiment 5 are between 96.3% and 105.1%, indicating that the formula of reagent R1 in Embodiment 1 and Embodiment 5 and the kits thereof have better repeatability and accuracy.

[0219] Application Example 2 Clinical Sample Comparison

[0220] The reagents prepared according to Example 1 and Example 5 were tested using a Hitachi 7180 fully automatic biochemical analyzer; wherein the comparative example was the same as Example 1 or 5 except that R1 did not contain acetylsalicylic acid, cimetidine and erythromycin.

[0221] The test wavelength is 546nm, the sample volume or calibration product is 9μL, then 180μL of R1 reagent is added, incubated at 37℃ for 5min, then 80μL of R2 reagent is added, and the absorbance A1 value is read after 20S, and the absorbance A2 is read after 4min and 42s of incubation at 37℃, then the absorbance ΔA=A2-A1;

[0222] Use standard products for multi-point calibration and use spline function for calculation to obtain the calibration curve, such as Figure 1 A and Figure 1 C. By Figure 1 It can be seen that the values ​​of each point of the calibrator are on the fitting line, the fit is good, and it can be used for the next sample test.

[0223] The concentration of valproic acid in the sample can be obtained by comparing its absorbance change with the standard curve. The detection results are compared with the results of HPLC-MS / MS method. The results are shown in Table 21.

[0224] HPLC-MS / MS method

[0225] Telmisartan was used as the internal standard. The chromatographic column conditions were: Agilent ZORBAX300SB-C18 column (2.1 mm × 150 mm, 5 μm); mobile phase: acetonitrile -20 mmol.L -1 Ammonium acetate (55:45); flow rate: 0.25 mL.min -1 , injection volume 5μL, autosampler temperature maintained at 4℃. Mass spectrometry conditions: negative ion mode detection, multiple reaction monitoring (MRM) scanning, scanning results: sodium valproate m / z143.0→m / z143.0, telmisartan m / z513.5→m / z469.4. Curtain gas (CUR) 25.00; collision gas (CAD) 6.00; GASI 40mL.min -1 ;GAS2: 60mL.min -1 , spray voltage (IS) 5500V; heating temperature 500℃; declustering voltage (DP) is -95V for sodium valproate and -134V for telmisartan; collision induced dissociation voltage (CE) is -5eV for sodium valproate and -29eV for telmisartan; collision chamber injection voltage (EP) is -10V for sodium valproate and -10V for telmisartan; and extraction voltage (CXP) is -10V for sodium valproate and -10V for telmisartan.

[0226] Sample treatment: Take 200 μL of sample and transfer it to a 2 mL centrifuge tube. Add 20 μL of internal standard solution (4 μg / mL) and mix well. Add 0.6 mL of acetonitrile and vortex mix for 10 seconds. Centrifuge at high speed (13000 r.min) -1 ) for 10 min, and take the supernatant for determination.

[0227] Table 21 Comparison of the values ​​measured by different kits and HPLC-MS / MS

[0228]

[0229] The commercial kits in Table 21 are from: Sekisui Medical Technology (China) Co., Ltd.; valproic acid detection kit (latex agglutination turbidimetry) Nanopia TDM Valproic Acid.

[0230] According to Table 21, combined Figure 2 The results of A, B, C, and D show that: From the comparison result dot plot, it can be seen that R1 does not contain acetylsalicylic acid or cimetidine and erythromycin, and the correlation between the measured values ​​and the HPLC-MS / MS measured values ​​is the worst. 2 =0.8994 (e.g. Figure 2 A in R1); the correlation between the measured values ​​of acetylsalicylic acid or cimetidine and erythromycin and the measured values ​​of HPLC-MS / MS is improved (such as Figure 2 B, C); from Figure 2 As can be seen from B, C, and D in the figure, the correlation R between the kit prepared by the present invention and the commercial kit in the HPLC-MS / MS determination value is 2 >0.975, but from the measurement results, the kit prepared by the present invention is closer to the commercial kit in terms of HPLC-MS / MS measurement values, which is better than the existing commercial kit.

[0231] The sample comparison results show that if the kit prepared by the present invention contains a drug that competes with the protein for binding, it has a good sample correlation with the HPLC-MS / MS measurement value, and the measurement values ​​are relatively close. Although the commercial kit has a good correlation, the overall value is low. The kit prepared by the present invention is closer to the commercial kit than the HPLC-MS / MS measurement value, and can provide a more accurate reference value for clinical use based on convenient detection.

[0232] The present invention further studies and finds that the plasma protein binding amount of the drug is affected by the drug concentration, the quality and quantity of plasma proteins and the dissociation constant. Each drug is different and the binding rate decreases with increasing dose. The reagent R1 (containing the dissociated component) contains 1-2 mg / mL of acetylsalicylic acid, or contains cimetidine (0.2-0.4 mg / mL) and erythromycin (0.1-0.2 mg / mL) at the same time. When valproic acid and acetylsalicylic acid are present at the same time, the competition for protein binding can change the plasma concentration of both. Acetylsalicylic acid competes with valproic acid for binding and increases the toxicity of valproic acid, that is, the plasma concentration of free valproic acid increases. When the sample is measured, the sample containing acetylsalicylic acid The R1 reagent is first combined with the sample and incubated in a 37°C water bath for 5 minutes. The valproic acid in the sample that is bound to plasma proteins will be competitively bound by acetylsalicylic acid, and the plasma concentration of free valproic acid will increase, making the measurement of the sample more accurate and not affected by interfering substances (such as hemoglobin). Reagent R1 (containing dissociation components) also contains cimetidine (0.2-0.4 mg / mL) and erythromycin (0.1-0.2 mg / mL), and the principle is similar to that containing acetylsalicylic acid.

Claims

1. A reagent R1 for detecting valproic acid by latex enhanced immunoturbidimetry, It is characterized in that The invention comprises a dissociator of protein-bound valproic acid, wherein the dissociator comprises 1) acetylsalicylic acid, or 2) cimetidine and erythromycin; the acetylsalicylic acid is 1-2 mg / mL, the cimetidine is 0.2-0.4 mg / mL, and the erythromycin is 0.1-0.2 mg / mL; The reagent R1 also includes: (1) a stabilizer, wherein the concentration of the stabilizer is 1-10%, and the stabilizer is chloride and / or disaccharide; the chloride is selected from one or more of sodium chloride, potassium chloride and magnesium chloride, and the disaccharide is selected from one or more of sucrose, maltose and trehalose; (2) a buffer, wherein the concentration of the buffer is 0.2-1%, and the buffer is selected from one or more of MES, Tris, MOPS and HEPES; (3) a surfactant, wherein the concentration of the surfactant is 0.01-0.1%, and the surfactant is a nonionic surfactant; the nonionic surfactant is a polyoxyethylene nonionic surfactant, a polyol nonionic surfactant, or an alkyl alcohol amide nonionic surfactant; (4) a metal ion chelator, wherein the concentration of the metal ion chelator is 0.1-1%, and the metal ion chelator is EDTA or its potassium salt; and, (5) a coagulant, wherein the concentration of the coagulant is 1-5%, the coagulant is a polysaccharide or polyvinyl pyrrolidone, and the polysaccharide is dextran; The above percentages are the mass volume percentages in reagent R1, in g / mL.

2. The reagent R1 as claimed in claim 1, It is characterized in that The sample for the test is from blood.

3. The reagent R1 as claimed in claim 1, It is characterized in that The sample for the test is from plasma or serum.

4. The reagent R1 as claimed in claim 1, It is characterized in that The content of the reagent R1 is: 2 mg / mL of acetylsalicylic acid, 0.35 mg / mL of cimetidine, and 0.2 mg / mL of erythromycin; The above percentages are the mass volume percentages in reagent R1, in g / mL.

5. The reagent R1 as claimed in claim 1, It is characterized in that The buffer is MES; and / or, The potassium salt of EDTA is EDTA-2K or EDTA-3K.

6. The reagent R1 as claimed in claim 5, It is characterized in that The chloride is sodium chloride or potassium chloride; the disaccharide is sucrose; the buffer is MES monohydrate; the surfactant is a polyol-type nonionic surfactant; the potassium salt of EDTA is EDTA-2K; and / or the dextran is a dextran with a molecular weight of 450,000-650,000.

7. The reagent R1 as claimed in claim 6, It is characterized in that The chloride is sodium chloride; and / or the surfactant is Tween.

8. The reagent R1 as claimed in claim 7, It is characterized in that The surfactant is Tween 20.

9. The reagent R1 as claimed in claim 8, It is characterized in that It further includes: (6) Valproic acid antigen; (7) a preservative, wherein the concentration of the preservative is 0.01-0.1%; and / or (8) a blocking agent, wherein the concentration of the blocking agent is 0.01-0.1%; The above percentages are the mass volume percentages in reagent R1, in g / mL.

10. Reagent R1 as claimed in claim 9, It is characterized in that The concentration of the valproic acid antigen is 130-150 ng / mL; the preservative is selected from one or more of gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3(2H)-isothiazolone 5-bromo-5-nitro-1,3-dioxane and Proclin-300; and / or the blocking agent is BSA or ethanolamine.

11. The reagent R1 as claimed in claim 10, It is characterized in that The preservative is sodium azide; and / or the blocking agent is ethanolamine.

12. The reagent R1 according to claim 11, It is characterized in that The sodium chloride is 1-5%, the sucrose is 1-5%, the dextran is 1-5%, the MES monohydrate is 0.2-1%, the Tween 20 is 0.01-0.1%, the EDTA-2K is 0.1-1%, the sodium azide is 0.01-0.1%, the ethanolamine is 0.01-0.1%, and the valproic acid antigen is 130-150 ng / mL, and the pH of the reagent R1 is 5-9; The above percentages are the mass volume percentages in reagent R1, in g / mL.

13. The reagent R1 as claimed in claim 12, It is characterized in that The reagent R1 comprises: 1-2 mg / mL acetylsalicylic acid, 1-3% sodium chloride, 1-3% sucrose, 2.5-3% dextran, 0.3-0.6% MES monohydrate, 0.02-0.05% Tween 20, 0.3-0.5% EDTA-2K, 0.03-0.05% sodium azide, 0.01-0.05% ethanolamine, and 140-145 ng / mL valproic acid antigen, pH 6-7; or, 0.2-0.4 mg / mL cimetidine, 0.1-0.2 mg / mL erythromycin, 1-3% sodium chloride, 1-3% sucrose, 2.5-3% dextran, 0.3-0.6% MES monohydrate, 0.02-0.05% Tween 20, 0.3-0.5% EDTA-2K, 0.03-0.05% sodium azide, 0.01-0.05% ethanolamine, and 140-145 ng / mL valproic acid antigen, pH 6-7; The above percentages are the mass volume percentages in reagent R1, in g / mL.

14. The reagent R1 according to claim 13, It is characterized in that The reagent R1 comprises: 2 mg / mL acetylsalicylic acid, 1% sodium chloride, 1% sucrose, 2.5% dextran, 0.533% MES monohydrate, 0.02% Tween 20, 0.5% EDTA-2K, 0.05% sodium azide, 0.05% ethanolamine, and 145 ng / mL valproic acid antigen, pH 6.8; or, 0.35 mg / mL cimetidine, 0.2 mg / mL erythromycin, 1% sodium chloride, 1% sucrose, 2.5% dextran, 0.533% MES monohydrate, 0.02% Tween 20, 0.5% EDTA-2K, 0.05% sodium azide, 0.05% ethanolamine, and 145 ng / mL valproic acid antigen, pH 6.8; The above percentages are the mass volume percentages in reagent R1, in g / mL.

15. A valproic acid detection kit, It is characterized in that It comprises the reagent R1 as described in any one of claims 1-14.

16. The valproic acid detection kit according to claim 15, It is characterized in that The valproic acid detection kit also includes a reagent R2, which contains latex microspheres, a protein stabilizer, a buffer, a surfactant, a metal ion chelator and / or a preservative.

17. The valproic acid detection kit according to claim 16, It is characterized in that The dosage of the latex microspheres is 5-20 μL / mL, the concentration of the protein stabilizer is 1-5%, the concentration of the buffer is 0.2-1%, the concentration of the surfactant is 0.001-0.01%, the concentration of the metal ion chelator is 0.1-0.5%, and the concentration of the preservative is 0.01-0.1%; The above percentages are the mass volume percentages in reagent R2, in g / mL.

18. The valproic acid detection kit according to claim 16, It is characterized in that The pH of the reagent R2 is 4-6, and the reagent R2 contains: The latex microspheres are valproic acid sensitized latex microspheres; The protein stabilizer is betaine hydrochloride; The buffer is one or more of MES, Tris, MOPS, and HEPES; The surfactant is a nonionic surfactant; The metal ion chelating agent is EDTA or its potassium salt; The preservative is one or more of gentamicin sulfate, sodium azide, 2-hydroxypyridine-N-oxide, chloroacetamide, imidazolidinyl urea, thimerosal, 2-methyl-3(2H)-isothiazolone, 5-bromo-5-nitro-1,3-dioxane and Proclin-300.

19. The valproic acid detection kit according to claim 18, It is characterized in that The protein stabilizer is betaine hydrochloride; the buffer is HEPES; the surfactant is a polyoxyethylene nonionic surfactant, a polyol nonionic surfactant or an alkyl alcohol amide nonionic surfactant; the potassium salt of EDTA is EDTA-2K or EDTA-3K; and / or the preservative is sodium azide.

20. The valproic acid detection kit according to claim 19, It is characterized in that The surfactant is a polyol type nonionic surfactant; and / or the potassium salt of EDTA is EDTA-2K.

21. The valproic acid detection kit according to claim 20, It is characterized in that The surfactant is Tween.

22. The valproic acid detection kit according to claim 21, It is characterized in that The surfactant is Tween 20.

23. The valproic acid detection kit according to claim 22, It is characterized in that The pH of the reagent R2 is 5-5.5, The dosage of the valproic acid sensitized latex microspheres is 5-10 μL / mL, the betaine hydrochloride is 1-5%, the HEPES is 0.2-1%, the Tween 20 is 0.001-0.01%, the EDTA-2K is 0.1-0.5%, the sodium azide is 0.01-0.1%, and the pH is 7-9; The above percentages are the mass volume percentages in reagent R2, in g / mL.

24. The valproic acid detection kit according to claim 23, It is characterized in that The pH of the reagent R2 is 5.5, the dosage of the valproic acid sensitized latex microspheres is 10 μL / mL, the betaine hydrochloride is 2%, the HEPES is 0.596%, the Tween 20 is 0.005%, the EDTA-2K is 0.3%, the sodium azide is 0.05%, and the pH is 8; The above percentages are the mass volume percentages in reagent R2, in g / mL.

25. The valproic acid detection kit according to any one of claims 18 to 24, It is characterized in that The preparation method of the valproic acid sensitized latex microspheres comprises: (1) Mix buffer B and latex microspheres, add EDC and NHS, shake for 20-50 minutes, add surfactant A, react for 10-30 minutes, and sonicate 10 times; (2) Add valproic acid monoclonal antibody and perform cross-linking reaction.

26. The valproic acid detection kit according to claim 25, It is characterized in that The shaking time is 35 minutes; and / or, the reaction time is 20 minutes.

27. The valproic acid detection kit according to claim 25, It is characterized in that It also includes adding a blocking agent to the reagent R1 for blocking; The buffer B is NaHCO 3 solution, the solid content of the latex microspheres is 5%, the surfactant A is poloxamer 188, the dosage ratio of EDC to latex microspheres is 0.5 μg / μL, the weight ratio of EDC to NHS is 1:10, the dosage ratio of poloxamer 188 to latex microspheres is 100-500 μg / μL, and the dosage ratio of valproic acid monoclonal antibody to latex microspheres is 0.5-2.5 μg / μL; The cross-linking reaction was performed by shaking for 20 minutes, ultrasonicating for 10 times, shaking for 60 minutes, ultrasonicating for 10 times, and finally shaking for 30 minutes.

28. A reagent for detecting valproic acid, It is characterized in that The method comprises the reagent R1 for detecting valproic acid by latex-enhanced immunoturbidimetry as described in any one of claims 1 to 14 and a reagent A, wherein the reagent A is a valproic acid standard and / or a valproic acid antibody.

29. The reagent according to claim 28, It is characterized in that The detection method is latex enhanced immunoturbidimetry; and / or the detection sample is from blood.

30. The reagent according to claim 29, It is characterized in that The sample for the test is from plasma or serum.

31. Use of the reagent R1 for detecting valproic acid by latex enhanced immunoturbidimetry as described in any one of claims 1 to 14 in detecting valproic acid.

32. The use according to claim 31, It is characterized in that The sample for detection is from blood; and / or, the detection method is latex enhanced immunoturbidimetry.

33. The use according to claim 32, It is characterized in that The sample for the test is from plasma or serum.

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