Valproic acid test kit

Abstract

The application discloses a valproic acid detection kit. Specifically, the application relates to a valproic acid derivative and a preparation method thereof, and a kit containing the valproic acid derivative and a preparation method thereof. The technical scheme of the application uses a novel valproic acid derivative and a coupling method of maleimide-sulfhydryl with high selectivity, so that the derivative and an enzyme are coupled in a 1:1 mode, and batch difference formed in a conventional coupling process is greatly reduced. The valproic acid detection kit is simple, fast and low in cost, and can be automatically detected on various mainstream machines.

Description

[0001] This application is a divisional application of Chinese patent application No. 201911042028.0, filed on October 30, 2019, entitled "Valproic acid derivatives and their use in immunoassay". Technical Field

[0002] This application relates to a valproic acid derivative and its preparation method, and a homogeneous enzyme immunoassay kit for valproic acid and its preparation method. Background Technology

[0003] The structural formula of valproic acid is shown below:

[0004]

[0005] Valproic acid is a medication used to treat epilepsy. The drug consists of sodium valproate and valproic acid, and can be used as monotherapy or as adjunctive therapy. Valproic acid is used to treat generalized epilepsy, including absence seizures, myoclonic seizures, and tonic-clonic seizures. For partial epilepsy, it is suitable for simple partial seizures, complex partial seizures, and partial-onset generalized seizures. In addition to its antiepileptic uses, it can also be used to treat febrile seizures, movement disorders, chorea, porphyria, schizophrenia, pain caused by herpes zoster, adrenal insufficiency, and to prevent alcohol withdrawal syndrome.

[0006] Common adverse reactions to valproic acid therapy include: gastrointestinal symptoms and changes in the menstrual cycle; less common adverse reactions include hair loss, constipation, drowsiness, dizziness, fatigue, headache, ataxia, mild tremor, abnormal excitement, restlessness, and irritability; with long-term use, pancreatitis, liver damage, acute liver necrosis, and thrombocytopenia may occasionally occur; allergic reactions may occasionally occur; and hearing loss and reversible hearing damage may occasionally occur.

[0007] Therefore, monitoring for adverse reactions to this drug is crucial during treatment. Due to individual differences in drug metabolism, clinical use should incorporate blood drug concentration monitoring to develop a reasonable dosing regimen and minimize the occurrence of adverse reactions.

[0008] Currently, there are various methods for monitoring valproic acid blood concentrations in clinical practice, such as high-performance liquid chromatography (HPLC), chemiluminescence immunoassay, homogeneous enzyme immunoassay, and latex agglutination turbidimetry. HPLC is time-consuming and difficult to automate; direct chemiluminescence immunoassay has high sensitivity but requires expensive chemiluminescence instruments, is cumbersome to operate, has a long detection time, low automation, and poor repeatability. Existing homogeneous enzyme immunoassay and latex agglutination turbidimetry also have limitations in application due to poor repeatability and linearity.

[0009] Patent application CN102507917A discloses a valproic acid enzyme-labeled conjugate, which is prepared by first activating the valproic acid derivative by adding tributylamine and isobutyl chloroformate to a solution of the valproic acid derivative; then conjugating it with glucose-6-phosphate dehydrogenase (G6PDH) at -2 to -8°C, purifying it using a gel chromatography column, and finally diluting the valproic acid enzyme-labeled conjugate with a buffer solution.

[0010] Patent application CN103242445A discloses a method for preparing enzyme-labeled conjugates: glucose-6-phosphate dehydrogenase is weighed and dissolved in phosphate buffer at room temperature to a final concentration of 3-5 mg / mL; valproic acid derivative is dissolved in dimethylformamide, activated by the tributylamine method, and cross-linked with glucose-6-phosphate dehydrogenase solution; after purification and dialysis, glucose-6-phosphate dehydrogenase-valproic acid conjugate is obtained.

[0011] Patent application CN108956971A discloses a valproic acid immunoassay reagent and its preparation and detection method, which involves dissolving valproic acid, 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide, and N-hydroxythiosuccinimide in morpholine ethanesulfonic acid (MES) solution for activation, and then coupling them with an enzyme.

[0012] All three routes described above rely on activating the reactive groups originally carried by valproic acid before reacting with the enzyme. Such strategies struggle to guarantee directional reactions and achieve 1:1 coupling between the derivative and the enzyme, leading to unstable batch-to-batch precision. Even under optimal conditions, reagents prepared using these strategies show reported intra-batch and inter-batch precision CVs of only around 4.7%.

[0013] Therefore, there is still a need in the art for an improved valproic acid derivative and an improved coupling method. Summary of the Invention

[0014] According to some embodiments, a valproic acid derivative is provided having the structure shown in Formula I:

[0015]

[0016] in

[0017] n is an integer from 1 to 10, preferably an integer from 2 to 6;

[0018] m is an integer from 1 to 10, preferably an integer from 1 to 5.

[0019] In some specific embodiments, the valproic acid derivative has the structure shown in Formula II:

[0020]

[0021] According to some embodiments, an enzyme label is provided comprising a valproic acid derivative according to this application and an enzyme. In some specific embodiments, the enzyme and the valproic acid derivative are covalently bound. In some specific embodiments, the enzyme is glucose hexaphosphate dehydrogenase.

[0022] According to some embodiments, a glucose hexaphosphate dehydrogenase variant is provided, in which the proline residue at position 254 is replaced by a cysteine ​​residue compared to the wild-type glucose hexaphosphate dehydrogenase. It should be understood that the proline residue at position 254 and its equivalent positions are included within the scope, depending on the species to which the glucose hexaphosphate dehydrogenase belongs.

[0023] In a specific implementation plan, the species of glucose hexaphosphate dehydrogenase can be selected from Leuconostoc mesenteroides.

[0024] According to some embodiments, a reagent is provided that comprises a valproic acid derivative according to this application.

[0025] According to other embodiments, a reagent is provided that comprises an enzyme label according to this application.

[0026] According to some embodiments, a method for preparing an enzyme label is provided, which includes the following steps:

[0027] 1) Provide a valproic acid derivative according to the present application, for example, provide a valproic acid derivative in an aprotic solvent;

[0028] 2) Provide the enzyme, for example, by providing the enzyme in a buffer solution;

[0029] 3) At 18°C ​​to 28°C, the valproic acid derivative and the enzyme are contacted for 1 hour to 4 hours (e.g., 1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or any value in between) at a molar ratio of 1:1 to 1:200 (preferably 1:50) to obtain an enzyme-labeled product.

[0030] In some specific implementations, the prepared enzyme label may be purified as needed, for example, but not limited to molecular sieve chromatography.

[0031] In the context of this application, the step number should not be interpreted as the order of operations.

[0032] In some specific implementations, steps 1) and 2) can be interchanged.

[0033] In some embodiments, the enzyme is provided in a buffer solution selected from PBS, Tris, TAPS, and TAPSO, wherein the buffer solution has a pH of 6.0 to 8.0.

[0034] In some implementations, an aprotic solvent provides the reaction medium environment, selected from one or a combination of acetonitrile, dimethylformamide, and dimethyl sulfoxide.

[0035] In some embodiments, the enzyme is glucose hexaphosphate dehydrogenase; particularly prior to coupling, the enzyme contains at least one free sulfhydryl group to allow for a directed and controlled coupling reaction.

[0036] In a specific implementation plan, a method for preparing an enzyme marker is provided, including the following steps:

[0037] 1) Dissolve the valproic acid derivative in N,N-dimethylformamide;

[0038] 2) Dissolve glucose hexaphosphate dehydrogenase containing one free sulfhydryl group in a buffer solution;

[0039] 3) Mix the solutions from steps 1) and 2) and contact them at 18°C ​​to 25°C (allowing for thorough mixing, such as moderate shaking) for 2 to 3 hours;

[0040] 4) Perform molecular sieve chromatography on the reaction product obtained in step 3) to obtain purified glucose hexaphosphate dehydrogenase-labeled valproic acid derivative.

[0041] According to some implementation schemes, enzyme markers prepared according to the methods of this application are provided.

[0042] According to some implementation schemes, a valproic acid detection kit is provided, comprising:

[0043] - The first reagent contains an anti-valproic acid antibody derived from: mice, rats, primates, sheep, poultry, humans, rabbits, horses, cattle, and camels; the anti-valproic acid antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments;

[0044] - A second reagent comprising an enzyme marker according to this application;

[0045] -Optional, quality control products; and / or

[0046] -Optional, calibrator.

[0047] In some implementations, the quality control material contains 30 μg / ml to 120 μg / ml valproic acid, such as 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 μg / ml (or any value between these values).

[0048] In some implementations, the calibrator contains 0 μg / ml to 150 μg / ml valproic acid, such as 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μg / ml (or any value between these values).

[0049] In some specific implementations, the valproic acid detection kit comprises:

[0050] -The first reagent, which comprises:

[0051] 30mM to 300mM buffer solution

[0052] 5mM to 20mM glucose-6-phosphate,

[0053] 5mM to 20mM oxidized β-nicotinamide adenine dinucleotide,

[0054] 0.01 μg / ml to 10 μg / ml anti-valproic acid monoclonal antibody,

[0055] 0.1 g / L to 5 g / L stabilizer

[0056] 0.1 g / L to 5 g / L surfactant,

[0057] 0.1 g / L to 5 g / L of preservatives;

[0058] -The second reagent, which comprises:

[0059] 30mM to 300mM buffer solution

[0060] The enzyme-labeled product of this application is available in concentrations from 0.01 μg / ml to 10 μg / ml.

[0061] 0.1 g / L to 5 g / L stabilizer

[0062] 0.1 g / L to 5 g / L surfactant,

[0063] 0.1 g / L to 5 g / L of preservatives;

[0064] According to some implementation schemes, the use of the valproic acid derivatives of this application in the preparation of detection devices is provided.

[0065] According to some implementation schemes, the use of the enzyme markers of this application in the preparation of detection devices is provided.

[0066] According to some implementation schemes, the use of the glucose hexaphosphate dehydrogenase variant of this application in the preparation of a detection device is provided.

[0067] In some implementations, the detection device is embodied in the form of reagents, kits, well plates, particles, chips, and test strips. Attached Figure Description

[0068] Figure 1 This is a synthetic route diagram for valproic acid derivatives.

[0069] Figure 2 This is a correlation analysis graph of the reagent kit in this application.

[0070] Figure 3 The difference in absorbance between batches of the reagent and the control reagent in this application. Detailed Implementation

[0071] The following specific examples illustrate this application in detail. The examples only describe in detail the synthesis method of the valproic acid derivative listed in the figure below, and the specific method of coupling the derivative with glucose hexaphosphate dehydrogenase.

[0072] Example 1. Synthesis and structural confirmation of valproic acid derivatives

[0073] according to Figure 1 The synthetic route for the valproic acid derivative is shown below, and the compound represented by Formula II is synthesized as follows:

[0074]

[0075] 1. Synthesis of Compound 3

[0076] Compound 1 (100 mg, 0.69 mmol) and compound 2 (150 mg, 0.69 mmol) were dissolved in DCM (15 mL), and triethylamine (209 mg, 2.07 mmol) and HATU (314 mg, 0.83 mmol) were added. The mixture was stirred at room temperature (18 to 25 °C) for 6 h. The solvent was removed under reduced pressure, and the mixture was purified by column chromatography to give compound 3 (150 mg, 64% yield) as a white solid.

[0077] 2. Synthesis of Compound 4

[0078] Compound 3 (150 mg, 0.46 mmol) was dissolved in methanol (10 mL), and concentrated HCl (2 mL) was added. The mixture was stirred at room temperature (18 to 25 °C) for 2 h. Water was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The aqueous phase was adjusted to pH 10 with NaOH (1 N), extracted with ethyl acetate, and the solvent was removed under reduced pressure to give compound 4 (128 mg, 100%).

[0079] 3. Synthesis of valproic acid derivatives

[0080] Compound 4 (50 mg, 0.18 mmol) and compound 5 (38 mg, 0.18 mmol) were dissolved in DCM (8 mL), and triethylamine (55 mg, 0.54 mmol) was added dropwise, followed by HATU (82 mg, 0.22 mmol). The mixture was stirred at room temperature (18 to 25 °C) for 16 h. After preparative purification, a white solid valproic acid derivative (55 mg, 71% yield) was obtained.

[0081] 4. Mass spectrometry (measured M) +1 525.5, M +23 574.6) and NMR ( 1 H-NMR analysis confirmed that the structure of the valproic acid derivative was correct.

[0082] Example 2. Method for preparing enzyme-labeled compounds (method of this application)

[0083] 1. The valproic acid derivative obtained in Example 1 was dissolved in N,N-dimethylformamide (10 mg / ml);

[0084] 2. Add 200 μl of glucose hexaphosphate dehydrogenase (derived from Leuconostoc mesenteroides) (artificially modified to contain a single free sulfhydryl group, i.e., proline at position 254 is replaced with cysteine ​​to obtain a free sulfhydryl group) solution (6.4 mg / ml, 0.2 M phosphate buffer, pH 8.0) to 750 μl of buffer solution (0.05 M Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH 7.2);

[0085] 3. Then add 50 μl of an N,N-dimethylformamide solution containing valproic acid derivatives;

[0086] 4. The above mixed solution should be thoroughly shaken at room temperature (18 to 28°C) for 2-3 hours;

[0087] 5. Molecular sieve chromatography was used to obtain valproic acid-labeled glucose hexaphosphate dehydrogenase (concentration 0.1 mg / mL-2.0 mg / mL).

[0088] Example 3. Method for preparing enzyme-labeled products (control method, coupling reaction of the carboxyl group of activated valproic acid with the amino group on G6PDH)

[0089] 1. Dissolve 15 mg of glucose-6-phosphate dehydrogenase in 12 mL of Tris buffer, then add 225 mg of reduced nicotinamide adenine dinucleotide (NADH), 135 mg of glucose-6-phosphate, 0.75 mL of carbitol, and 2.25 mL of dimethyl sulfoxide in sequence; the pH of the Tris buffer is 9.0, and the concentrations of each component are: 0.05 mol / L Tris, 3.3 mmol / L magnesium chloride, and 145.4 mmol / L sodium chloride.

[0090] 2. Activation of valproic acid derivative: 10 mg of valproic acid derivative was dissolved in 420 μL of dimethyl sulfoxide and 180 μL of dimethylformamide, and 6 μL of tributylamine and 350 μL of isobutyl chloroformate were added. The mixture was stirred at 2-8 °C for 30 minutes.

[0091] 3. Mix the solutions obtained in steps 1 and 2, stir at 2 to 8°C for 12 to 16 hours, and purify the conjugated enzyme-labeled antigen by G-25 gel chromatography to obtain glucose-6-phosphate dehydrogenase-labeled valproic acid derivative.

[0092] Example 4. Preparation of reagents

[0093] 1. Preparation of the first reagent:

[0094]

[0095]

[0096] 2. Preparation of the second reagent:

[0097]

[0098] 3. Quality control products and calibrators:

[0099] The inherent properties and effects of the reagents in this application do not depend on quality control products and calibrators; self-made or commercially available products can be used.

[0100] The quality control products are pure valproic acid diluted with buffer solution, with concentrations of 30-40 μg / ml, 70-80 μg / ml, and 115-125 μg / ml, respectively.

[0101] The calibrators are pure valproic acid diluted with buffer solution, with concentrations of 0 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml, and 150 μg / ml.

[0102] 4. Reagent kit assembly:

[0103] Assemble the above reagents (optionally including quality control and calibrators) into a homogeneous enzyme immunoassay kit for valproic acid.

[0104] Example 5. Performance Experiment of the Homogeneous Enzyme Immunoassay Kit for Valproic Acid

[0105] 1. The reagent kit in this application is based on a competitive reaction, and the detection principle is as follows:

[0106] In a homogeneous reaction system, valproic acid and glucose-6-phosphate dehydrogenase-valproic acid conjugate in the sample simultaneously compete for binding sites to anti-valproic acid antibodies. Since enzyme activity decreases after the antibody binds to the conjugate, the more free valproic acid in the sample, the more antibody binding sites there are, and the less antibody binds to the enzyme label. The unbound enzyme label catalyzes the oxidation of β-nicotinamide adenine dinucleotide (NAD). + Valproic acid is converted into β-nicotinamide adenine dinucleotide (NADH). The concentration of valproic acid in the sample is directly proportional to the amount of NADH generated. The concentration of valproic acid in the sample can be obtained by observing the change in absorbance.

[0107] 2. Biochemical analyzer parameters

[0108] Table 1. Parameters of the valproic acid reagent kit (Hitachi 7180) of this application

[0109] Analysis points [Rate-A]

[10]

[19]

[24] WAVE(SUB / MAIN)

[410]

[340] S.VIL. [2.0] S.R1

[180] S.R3

[60] ABS.LIMIT:

[32000] [Incrementing] CALIB type [Logit-Log4p] POINT parameters [6]SPAN POINT[6] Calibrator 0.0, 10.0, 25.0, 50.0, 100.0, 150.0μg / ml sample Various physiological samples (such as serum, plasma, saliva, whole blood, urine, etc.)

[0110] 3. Repeatability experiment

[0111] Repeatability tests were performed using the calibration curve established above, with high, medium, and low quality control samples tested 20 times each.

[0112] Table 2. Repeatability test results of the valproic acid reagent kit of this application

[0113] Test Quality Control 1 Quality Control 2 Quality Control 3 1 28.1 76.6 119.7 2 29.0 77.3 123.0 3 28.2 76.7 119.6 4 28.0 78.3 131.0 5 28.1 79.2 119.1 6 28.1 80.3 127.0 7 27.9 75.1 124.2 8 27.5 76.6 123.9 9 28.0 79.5 121.7 10 27.5 77.8 126.1 11 27.9 77.5 127.0 12 26.7 78.4 122.9 13 27.6 76.7 126.8 14 27.5 76.7 121.8 15 27.4 77.8 124.3 16 26.5 78.7 119.5 17 27.7 77.2 122.8 18 27.4 74.4 126.4 19 26.9 78.1 121.8 20 27.1 78.0 119.6 mean 27.7 77.5 123.4 Standard deviation 0.580 1.40 3.23 CV 2.10％ 1.81％ 2.62％

[0114] As shown in Table 1, the sample test was repeated 20 times, and the CV was less than 2.6%.

[0115] 4. Accuracy Experiment

[0116] United States Pharmacopeia (USP) pure products were dissolved in DMSO to stock solutions of different concentrations, and then diluted in serum at the same ratio (at least 20 times) to prepare serum VPA (variation partition analysis) solutions of different concentrations. The reagents were measured and the deviations from the theoretical values ​​were calculated.

[0117] Table 3. Accuracy test results of the valproic acid reagent kit of this application

[0118] USP Measured value 1 Measured value 2 Measured value 3 mean relative deviation absolute deviation 25 22.9 23.5 22.9 23.1 -7.6％ -1.9 30 27.4 27.5 28.0 27.6 -7.9％ -2.4 50 49.7 50.3 50.2 50.1 0.1％ 0.1 75 78.6 74.6 77.7 77.0 2.6％ 2.0 100 100.5 104.7 104.8 103.3 3.3％ 3.3 125 123.1 122.4 125.3 123.6 -1.1％ -1.4

[0119] 5. Drug Interference Experiment

[0120] Thirteen compounds and drugs were selected. When the concentration of valproic acid was around 90 μg / ml, the following concentrations of compounds did not show statistically significant interference.

[0121] Table 4. Anti-interference experiment

[0122]

[0123] 6. Linear Experiment

[0124] Low-value samples and high-value samples were diluted using an arithmetic dilution method, and each sample was tested three times. The obtained linear data are shown in Table 5. Figure 2 .

[0125] Table 5. Linearity data for the valproic acid reagent kit

[0126] Measured value 1 Measured value 2 Measured value 3 mean Theoretical value relative deviation absolute deviation 1 10.0 10.2 10.1 10.1 9.8 3.1％ 0.3 2 27.5 25.8 25.1 26.1 26.5 -1.4％ -0.4 3 41.6 44.7 43.1 43.1 43.2 -0.2％ -0.1 4 58.0 62.7 65.3 62.0 59.9 3.6％ 2.1 5 75.5 75.9 79.0 76.8 76.6 0.3％ 0.2 6 88.5 89.3 89.8 89.2 93.2 -4.3％ -4.0 7 108.4 108.2 110.7 109.1 109.9 -0.8％ -0.8 8 130.5 129.1 130.8 130.1 126.6 2.7％ 3.5 9 139.8 144.9 147.7 144.1 143.3 0.5％ 0.8 10 159.7 155.8 157.9 157.8 160.0 -1.4％ -2.2 11 172.9 180.9 179.7 177.8 176.7 0.6％ 1.1

[0127] 7. Batch-to-batch variation

[0128] Three batches of the reagents used in this application (Example 2) and the control reagent (Example 3) were used for calibration, and the differences in absorbance changes between different batches were calculated, as shown in Table 6. Figure 3 .

[0129] Table 6. Inter-batch variation data of reagents

[0130]

[0131] The advantage of this application lies in the use of a novel valproic acid derivative and a highly selective maleimide-thiol coupling method, which allows the derivative and enzyme to be coupled in a one-to-one ratio, greatly reducing the batch-to-batch variation caused by ordinary coupling processes.

[0132] The valproic acid reagent prepared by this method has good specificity and no significant cross-reactivity with 13 common drugs; the accuracy and precision are very high, with a detection CV of less than 2.6% and a recovery deviation of less than 8%. The valproic acid detection kit of this application is simple, fast, and low in cost, and can be used for automated detection on a variety of mainstream instruments.

Claims

1. A valproic acid detection kit comprising: - a first reagent comprising a buffer and an anti-valproic acid antibody: - a second reagent comprising a buffer and an enzyme label; wherein: the buffer in the first reagent and the second reagent is each independently selected from the group consisting of: TAPSO, phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer and HEPES buffer; the buffer pH in the first reagent and the second reagent is each independently from 5.0 to 8.5; the anti-valproic acid antibody is derived from any one of: mouse, rat, rabbit, camel, primate, horse, sheep, avian; the anti-valproic acid antibody is selected from the group consisting of: a monoclonal antibody or an antigen-binding fragment thereof, a polyclonal antibody or an antigen-binding fragment thereof; the enzyme label comprises or consists of: a valproic acid derivative and a glucose-6-phosphate dehydrogenase variant; the glucose-6-phosphate dehydrogenase variant and the valproic acid derivative are covalently bound; the valproic acid derivative is a structure represented by Formula I: Formula I wherein, n is an integer from 1 to 10; m is an integer from 1 to 10; In comparison to the wild-type Leuconostoc mesenteroides Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase variant has a proline residue at position 254 replaced with a cysteine residue.

2. The valproic acid detection kit according to claim 1, wherein: n is an integer from 2 to 6.

3. The valproic acid detection kit according to claim 1, wherein: m is an integer from 1 to 5.

4. The valproic acid detection kit according to claim 1, wherein: the pH is from 7.0 to 8.

0.

5. The valproic acid detection kit according to claim 1, wherein: the valproic acid derivative is covalently bound at the 254th amino acid residue of the glucose-6-phosphate dehydrogenase variant.

6. The valproic acid detection kit according to claim 1, further comprising: a quality control or calibration.

7. The valproic acid detection kit according to claim 1, wherein: - the first reagent comprises: 30 mM to 300 mM buffer, 5 mM to 20 mM glucose-6-phosphate, 5 mM to 20 mM oxidized β-nicotinamide adenine dinucleotide, 0.01 pg / ml to 10 pg / ml anti-valproic acid monoclonal antibody, 0.1 g / L to 5 g / L stabilizer, 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L preservative; - the second reagent comprises: 30 mM to 300 mM buffer, 0.01 pg / ml to 10 pg / ml of the enzyme label, 0.1 g / L to 5 g / L stabilizer, 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L preservative; wherein: the stabilizer in the first reagent and the second reagent is each independently selected from the group consisting of: bovine serum albumin, trehalose, sucrose, mannitol, glycerol, glycine and polyethylene glycol 6000; the preservative in the first reagent and the second reagent is each independently selected from the group consisting of: an azide compound, MIT, a biological preservative PC; the azide compound is sodium azide or lithium azide; the anti-valproic acid antibody is a murine antibody.

8. The valproic acid test kit according to claim 7, wherein: the biological preservative PC is PC-300.

9. The valproic acid test kit according to claim 6, wherein: the quality control comprises 30 μg / ml to 120 μg / ml valproic acid; the calibrator comprises 0 μg / ml to 150 μg / ml valproic acid.

10. The valproic acid test kit according to claim 1, wherein the valproic acid derivative is a structure shown in formula II: Formula II.

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