Phenytoin test kit

Abstract

This application relates to a phenytoin detection kit. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one mutation selected from the following compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, or G426C. The detection kit prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibits high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201911403882.5 (filed on December 31, 2019) entitled “6-phosphate dehydrogenase mutant and its use in the preparation of phenytoin detection reagent”. Technical Field

[0002] This application relates to the field of biological detection, and in particular to a multi-site mutated enzyme glucose-6-phosphate dehydrogenase (G6PDH) and its application in a phenytoin detection kit. Background Technology

[0003] Haptens are certain small molecules (molecular weight less than 4000 Da) that, when alone, cannot induce an immune response (i.e., they lack immunogenicity). However, when they cross-link or bind with large protein molecules or non-antigenic carriers such as polylysine, they acquire immunogenicity and induce an immune response. These small molecules can bind to response effect products and thus possess antigenicity. They only exhibit immunoreactivity but lack immunogenicity; they are also known as incomplete antigens.

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot independently stimulate the production of antibodies in humans or animals. It only has immunoreactivity, not immunogenicity, and is also called an incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and stimulate the animal to produce corresponding antibodies. Once a hapten binds to a protein, it forms an antigenic cluster of that protein. Some substances with smaller molecular weights than typical haptens but with chemically active groups of specific structures (such as penicillin and sulfonamides) are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites suitable for sandwich assays, therefore they cannot be detected using the double-antibody sandwich method; instead, a competitive assay is often employed. The principle is that the antigen in the sample and a certain amount of enzyme-labeled antigen compete for binding to the solid-phase antibody. The higher the antigen content in the sample, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This method is commonly used for the ELISA assays of small molecule hormones and drugs.

[0006] Phenytoin (PTN), as a specific example of a hapten, is an exogenous small molecule antiepileptic drug. This substance is not present in the serum or plasma of normal individuals; clinically, its sodium salt form is generally used.

[0007] Phenytoin sodium, as a traditional antiepileptic drug, primarily works by blocking voltage-dependent sodium... + Channels, reducing Na +Influx stabilizes neuronal cell membranes, reduces their excitability, and thus prevents localized abnormal discharges from spreading to normal brain tissue.

[0008] Phenytoin sodium reaches peak concentration 1.5 to 30 hours after oral administration, and effective blood concentrations are generally achieved with continuous administration for 6 to 10 days. It is inexpensive and effective, and is currently widely used in clinical practice. However, due to its unique pharmacokinetics and narrow therapeutic window, it is prone to overdose and poisoning, and the clinical manifestations of poisoning are diverse, making misdiagnosis easy. Therefore, continuous monitoring of phenytoin blood concentration is necessary during clinical diagnosis.

[0009] The optimal therapeutic effect is usually achieved at a blood concentration of 10-20 μg / ml without causing toxicity to the human body. However, blood concentrations above 20 μg / ml can cause liver and kidney toxicity, leading to coma in mild cases and death in severe cases. Generally, after discontinuing the medication for 2-4 weeks, the blood concentration of phenytoin can be reduced to 0.5 μg / ml or lower through metabolism by the liver and kidneys.

[0010] Currently known methods for phenytoin detection include enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay (HMI), chemiluminescence immunoassay (CIA), high-performance liquid chromatography (HPLC), and fluorescence polarization assay. However, these methods all have several drawbacks. For example, radioimmunoassay (RIA) suffers from radioactive contamination, short shelf life, and inconvenient operation. ELISA is cumbersome and time-consuming, making it unsuitable for clinical use. While CIA offers good sensitivity, it requires specialized equipment, resulting in high costs and hindering widespread adoption. In clinical diagnostic testing, homogeneous enzyme immunoassay (EMIT) is the primary method.

[0011] The principle of homogeneous enzyme immunoassay: In a homogeneous liquid reaction system, enzyme-labeled antigen (such as G6PDH-phenytoin) and unlabeled antigen (phenytoin) compete for binding with a quantitative antibody (phenytoin antibody). The more the antibody binds to the unlabeled antigen, the more activity of the enzyme-labeled antigen is released, and the more NADH is generated from the substrate NAD+ by the enzyme. By detecting the change in absorbance of NADH at a wavelength of 340 nm, the content of phenytoin in the liquid can be calculated. Summary of the Invention

[0012] In view of the needs of the art, this application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in the preparation of a phenytoin detection kit.

[0013] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. Unlike the published glucose-6-phosphate dehydrogenase mutant in US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose-6-phosphate dehydrogenase mutant of this application contains mutations selected from the following: D306C, G426C, and D375C.

[0014] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided, said glucose-6-phosphate dehydrogenase mutant being selected from the sequences shown below: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

[0015] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0016] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0017] According to some embodiments, a host cell is provided that contains the expression vector of this application. The host cell can be prokaryotic (such as bacteria) or eukaryotic (such as yeast).

[0018] According to some embodiments, a conjugate is provided, which is formed by conjugating the glucose-6-phosphate dehydrogenase mutant of this application with a hapten at a molar ratio of 1:n.

[0019] In some implementations, n is 1 to 50, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0020] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 1:1.

[0021] In some specific implementations, the molecular weight of the hapten is from 100 Da to 4000 Da, for example: 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1 000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

[0022] According to this application, those skilled in the art will understand that "hapten" also includes its derivative forms. To facilitate coupling with glucose-6-phosphate dehydrogenase, haptens that do not inherently possess a coupling group (e.g., a group that reacts with a thiol group) (e.g., phenytoin) can be modified to have a linker for covalent binding with a thiol group. Therefore, in this application, a hapten derivative refers to a hapten modified to possess a thiol-reactive group.

[0023] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0024] Haptens include, but are not limited to: phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides, phenolic acids, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic acid metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma glycopeptide, polyamines, histamine, thyroid-stimulating hormone, prolactin, placental lactogen, growth hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, inverse triiodothyronine, free thyroxine, free triiodothyronine. Amino acids, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfates, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, adrenaline, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0025] In the specific implementation plan, the hapten is phenytoin or its derivative.

[0026] In a specific embodiment, the hapten is a phenytoin derivative having a thiol reactive group, such as methyl sulfone, bromoacetyl, vinyl sulfone, or aziridine. In a specific embodiment, the hapten is a phenytoin derivative, as shown in Formula I:

[0027]

[0028] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0029] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of phenytoin detection reagents is provided.

[0030] According to some implementation schemes, the use of the conjugates of this application in the preparation of phenytoin detection reagents is provided.

[0031] In the specific implementation plan, the detection reagents are selected from: enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay (CIA) reagents, homogeneous enzyme immunoassay (HIA) reagents, and latex-enhanced immunoturbidimetric assay (LTIA) reagents.

[0032] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0033] According to some implementation schemes, a phenytoin detection kit is provided, comprising:

[0034] - A first reagent, comprising a substrate and a phenytoin antibody; the substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0035] - A second reagent, wherein the second reagent comprises the conjugate of this application;

[0036] -Optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 mg / L to 42 mg / L phenytoin; and

[0037] -Optionally, a quality control sample comprising 10 mM to 500 mM buffer solution and 0 mg / L to 42 mg / L phenytoin.

[0038] According to one embodiment, a phenytoin detection kit is provided, comprising:

[0039] The first reagent comprises:

[0040] 10mM to 500mM buffer solution

[0041] 5mM to 25mM substrate,

[0042] Phenytoin antibodies at concentrations ranging from 0.1 mg / L to 5 mg / L

[0043] 10mM to 300mM NaCl,

[0044] 0.1 g / L to 5 g / L stabilizer

[0045] 0.1 g / L to 5 g / L surfactant,

[0046] 0.1 g / L to 5 g / L of preservatives;

[0047] The second reagent comprises:

[0048] 10mM to 500mM buffer solution

[0049] 0.1 mg / L to 1 mg / L according to the conjugate of this application,

[0050] 0.1 g / L to 5 g / L stabilizer

[0051] 0.1 g / L to 5 g / L surfactant,

[0052] Preservatives ranging from 0.1 g / L to 5 g / L.

[0053] In some embodiments, the buffer solution is selected from one or a combination of the following: glycerol buffer, phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolpropane buffer; preferably, phosphate buffer; the concentration of the buffer solution is from 10 mmol / L to 500 mmol / L, preferably 100 mM; the pH of the buffer solution is 6-8, preferably 7.2 or 7.0.

[0054] In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; preferably bovine serum albumin.

[0055] In some embodiments, the surfactant is selected from one or a combination of the following: Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0056] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, PC-300, thimerosal; the azide is selected from: sodium azide, lithium azide.

[0057] In some embodiments, the substrate comprises: glucose-6-phosphate and β-nicotinamide adenine dinucleotide.

[0058] In some embodiments, a method for preparing a coupling compound is provided, comprising the steps of:

[0059] 1) Provide a phenytoin derivative, preferably provided in an aprotic solvent;

[0060] 2) Provide the defined glucose-6-phosphate dehydrogenase mutant, preferably provided in a buffer solution;

[0061] 3) The phenytoin derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at 18°C ​​to 28°C for 1 hour to 4 hours, preferably 2 hours to 3 hours, so that the phenytoin derivative and the glucose-6-phosphate dehydrogenase mutant are coupled to obtain the conjugate.

[0062] 4) Optionally, the conjugate is purified, preferably desalted;

[0063] Steps 1) and 2) can be interchanged or performed in parallel;

[0064] The buffer solution is selected from PBS, Tris, TAPS, and TAPSO, and the pH of the buffer solution is 6.0 to 8.0.

[0065] The aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide;

[0066] Preferably, prior to step 3), the glucose-6-phosphate dehydrogenase mutant contains a free sulfhydryl group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free sulfhydryl group at position 306, 375, or 426. Attached Figure Description

[0067] Figure 1 Structural diagram of phenytoin.

[0068] Figure 2 Structural diagram of phenytoin derivatives.

[0069] Figure 3A .G6PDH (wild type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0070] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0071] Figure 3C The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0072] Figure 3D The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0073] Example

[0074] Example 1. Synthesis of phenytoin derivatives

[0075] 1. Synthesis of Compound 2

[0076] Phenytoin (2.00 g, 7.94 mmol) and K2CO3 (1.65 g, 11.90 mmol) were dissolved in DMF (10 mL), and compound 1 (1.42 g, 6.35 mmol) was added to the reaction system. The mixture was heated to 50 °C and reacted for 16 h.

[0077] The reaction system was brought back to room temperature (20-25℃), water was added to the reaction system, and the mixture was extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure. The mixture was purified by chromatography (EA / PE = 1:3) to give a colorless oily compound 2 (2.04 g, 81.6%).

[0078] 2. Synthesis of Compound 3

[0079] Compound 2 (2.04 g, 5.20 mmol) was dissolved in ethanol (50 mL), and NaOH (2 N, 10 mL) was added to the reaction system. The mixture was stirred at room temperature (20-25 °C) for 2 h. The pH was adjusted to 5 with HCl (1 N), the solvent was removed under reduced pressure, and the mixture was purified by column chromatography (MeOH / DCM = 1:20) to give a white solid compound 3 (1.20 g, 60.0%).

[0080] 3. Synthesis of phenytoin derivatives

[0081] Compound 3 (117 mg, 0.32 mmol) and compound 4 (56 mg, 0.32 mmol) were dissolved in DCM (5 mL), and triethylamine (98 mg, 0.96 mmol) and HATU (147 mg, 0.39 mmol) were added dropwise. The mixture was stirred at room temperature (20-25 °C) for 5 h. Water (30 mL) was added to the reaction system, and the mixture was extracted with DCM. The organic phase was washed with saturated brine, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The mixture was then purified to obtain the phenytoin derivative (100 mg, 67%).

[0082]

[0083] The purpose of this embodiment is to give phenytoin a group that can bind to enzymes.

[0084] Example 2. Coupling of phenytoin derivatives with G6PDH molecules

[0085] According to the G6PDH-phenytoin conjugate of this application, the coupling is carried out in the following manner: the thiol reactive group (such as, but not limited to, maleimide group) on the phenytoin derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0086] 1. Solution preparation:

[0087] Phenytoin derivative solution: 10 mg / ml of the phenytoin derivative prepared in Example 1 was dissolved in DMF;

[0088] G6PDH solution: G6PDH (the mutant of this application or the prior art mutant), PB 100mmol, NaCl 100mmol, pH=8.0;

[0089] Coupling solution: 100mM PB / K, 100mM EDTA, 150mM NaCl, pH=7.2;

[0090] Desalination solution: 100mM PB / K, 100mM EDTA, 150mM NaCl, pH=7.2.

[0091] 2. Coupling procedure: 4 ml of G6PDH solution, 15.2 ml of coupling solution and 0.8 ml of phenytoin derivative solution were reacted at room temperature (20 to 25 °C) for 4 h.

[0092] 3. After shaking the above reaction system at room temperature for 4 hours, elute with the above desalting solution using a desalting column, collect the protein peak, and the obtained product is G6PDH-phenytoin conjugate.

[0093] Example 3. Preparation of the reagent kit

[0094] Prepare the following kit for detecting phenytoin, comprising:

[0095] Reagent R1 contains:

[0096] 100mM PB buffer, pH 7.2

[0097] 15mM glucose-6-phosphate

[0098] 15mM β-nicotinamide adenine dinucleotide

[0099] 2.5 mg / L phenytoin antibody (commercially available antibody, no special restrictions)

[0100] 150mM NaCl

[0101] 1 g / L bovine serum albumin

[0102] 1g / L Tween20

[0103] 1 g / L sodium azide;

[0104] Reagent R2 includes:

[0105] 100mM PB buffer, pH 7.2

[0106] 0.1 mg / L G6PDH-phenytoin conjugate

[0107] 1 g / L bovine serum albumin

[0108] 1g / L Tween 20

[0109] 1 g / L sodium azide;

[0110] Calibrator: 100mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 42 mg / L phenytoin (or add as needed);

[0111] Quality control: 100mM PB buffer, pH 7.2, and 5.0, 15, and 25 mg / L phenytoin (or add as needed).

[0112] Detection example

[0113] Reaction time: 10 min, including an incubation time of 4.7 min. After adding reagent R2 and incubating for 1 min, the absorbance A1 is measured and read. After incubating for another 1 min, the absorbance A2 is measured and read. Calculate ΔA = (A2 - A1) / min. Calculate the phenytoin content in the sample using the calibration curve: Phenytoin = Sample tube absorbance * Calibrator concentration / Calibrator absorbance.

[0114] The performance of the phenytoin detection kit prepared in Example 3 was tested. The main detection performance indicators were total imprecision, repeatability, recovery, linearity, and accelerated stability at 37°C.

[0115] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0116]

[0117]

[0118] Example 1. Calibration absorbance of phenytoin detection kit

[0119] Table 2. Calibration absorbance of phenytoin detection kit

[0120]

[0121] Note: In the prior art, the mutant designated A45C has a mutation site corresponding to... Figure 3A 46th place.

[0122] Example 2. Total imprecision of the phenytoin detection kit

[0123] Table 3. Total Imprecision

[0124] Example 3. Repeatability of Phenytoin Detection Kit

[0125] Table 4. Repeatability

[0126]

[0127] Example 4. Recovery of phenytoin detection kit

[0128] Table 5. Recycling

[0129]

[0130]

[0131] Example 5. Phenytoin Detection Kit Linearity

[0132] Table 6. Linear

[0133]

[0134] Test Example 6: Accelerated Stability at 37℃

[0135] Table 7. Accelerated stability at 37℃

[0136]

[0137] After acceleration at 37°C for 7 days, the calibrated absorbance of the reagent in this application decreased by about 15%, while the calibrated absorbance of the control reagent after acceleration at 37°C for 7 days was about 95%.

[0138] Example 7. Antibody inhibition rate

[0139] 1. Detection principle of antibody inhibition rate

[0140] When the antibody binds to the G6PDH-phenytoin conjugate, the steric hindrance affects the activity of the G6PDH enzyme, thereby reducing its efficiency in catalyzing the conversion of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental groups with and without the antibody can be compared. This difference reflects the inhibitory ability of the antibody on G6PDH.

[0141] 2. Reaction system

[0142] Table 8. Preparation of reagents for detecting antibody inhibition rate

[0143]

[0144] Table 9. Instrument parameters for antibody inhibition rate detection

[0145] Testing models Abbott C16000 Analysis / Time / Point Rate / 10min / 28-32 points R1 / S 120:20 Wavelength (sub- / primary) 405 / 340 reaction type Increasing

[0146] 3. Results

[0147] By comparing the absorbance values ​​of the G6PDH-phenytoin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0148] Antibody inhibition rate = [1 - (change in absorbance of G6PDH-phenytoin with antibody / change in absorbance of G6PDH-phenytoin without antibody)] × 100%.

[0149] Compared to previously published mutation sites, the mutants in this application show a significant improvement in antibody inhibition rate, reaching over 30% and up to 55%. In contrast, commonly used mutation sites (such as A45C and K55C) have inhibition rates of only around 40% or even lower.

[0150] Table 10. Antibody inhibition rates of different G6PDH mutants

[0151]

[0152] While not limited to specific theories, this can be partially explained as follows: Compared to existing G6PDH mutants, the mutation sites (i.e., the sites introducing free sulfhydryl groups) in the enzyme mutants of this application (D306C, D375C, G426C) are the locations where haptens (such as hormones, small molecule drugs, etc.) couple. When a hapten binds to a hapten-specific antibody at this site, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme, while the introduction of the mutation does not substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is crucial, requiring consideration of G6PDH enzyme activity, the spatial folding of the coupled molecule, and sufficient exposure of the hapten epitope.

[0153] Since the enzyme mutant has a significant increase in antibody inhibition rate, the reagent kit prepared by conjugating the enzyme mutant with phenytoin has a significant improvement in repeatability, total imprecision, linearity, and stability.

Claims

1. A phenytoin detection kit, comprising: - First reagent, the first reagent comprising a substrate and phenytoin antibody; - A second reagent, wherein the second reagent contains a conjugate; The conjugate is formed by coupling a glucose-6-phosphate dehydrogenase mutant with a phenytoin derivative in a molar ratio of 1:

1. The phenytoin derivative is shown in Formula I: Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains the mutant D306C; the glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No.

2.

2. The phenytoin detection kit according to claim 1, further comprising any one or a combination of the following: The calibrator contains 10 mM to 500 mM buffer solution and 0 mg / L to 42 mg / L phenytoin; Quality control material, wherein the quality control material comprises 10 mM to 500 mM buffer solution and 0 mg / L to 42 mg / L phenytoin.

3. The phenytoin detection kit according to claim 1, comprising: The first reagent comprises: 10mM to 500mM buffer solution 5mM to 25mM substrate, 0.1 mg / L to 5 mg / L phenytoin antibody, 10mM to 300mM NaCl, 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 of preservatives; The second reagent comprises: 10mM to 500mM buffer solution The conjugate, at concentrations of 0.1 mg / L to 1 mg / L 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 of preservatives; The buffer solutions in the first and second reagents are each independently selected from one or a combination of the following: glycerol buffer, phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer; the pH of the buffer solutions is 6 to 8; The stabilizers in the first reagent and the second reagent are each independently selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; The surfactants in the first reagent and the second reagent are each independently selected from one or a combination of the following: Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, and AEO7; The preservatives in the first reagent and the second reagent are each independently selected from one or a combination of the following: azide, MIT, PC biological preservative, thimerosal; The azide is selected from: sodium azide and lithium azide; The substrate comprises: glucose-6-phosphate and β-nicotinamide adenine dinucleotide.

4. The phenytoin detection kit according to claim 3, comprising: The first reagent comprises: 100mM to 300mM buffer solution 10mM to 20mM substrate, Phenytoin antibodies at concentrations of 1 mg / L to 2.5 mg / L 150mM to 200mM NaCl, 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives; The second reagent comprises: 100mM to 300mM buffer solution The conjugate, at concentrations of 0.1 mg / L to 1 mg / L 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives.

5. The phenytoin detection kit according to claim 1, comprising: a first reagent, comprising: 100mM PB buffer, pH 7.2 15mM glucose-6-phosphate 15mM β-nicotinamide adenine dinucleotide, 2.5 mg / L phenytoin antibody, 150mM NaCl, 1g / L bovine serum albumin, 1g / L Tween20 1 g / L sodium azide; The second reagent comprises: 100mM PB buffer, pH 7.2 The conjugate at 0.1 mg / L 1g / L bovine serum albumin, 1g / L Tween20 1 g / L sodium azide; Calibrators, comprising: 100mM PB buffer, pH 7.2, and 0 mg / L to 42 mg / L phenytoin; Quality control materials include: 100mM PB buffer, pH 7.2, and 5.0 mg / L to 25 mg / L phenytoin.

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