Use of conjugates in the preparation of phenytoin detection reagents

Abstract

This application relates to the use of conjugates in the preparation of phenytoin detection reagents. Specifically, the 6-glucose phosphate dehydrogenase mutant of the present application comprises one of the following mutations, D306C, D375C, and G426C, compared to wild-type 6-glucose phosphate dehydrogenase. The detection kit prepared using the 6-glucose phosphate dehydrogenase mutant of the present application has strong specificity, high sensitivity, convenient operation, short detection time, and accurate quantitative determination, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of Chinese patent application No. 2019114038825 (filing date December 31, 2019) "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Phenytoin Detection Reagents". Technical Field

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

[0003] Haptens are small molecules (molecular weight less than 4000 Da) that cannot induce an immune response on their own, meaning they are not immunogenic. However, when cross-linked or bound to a macromolecular protein or a non-antigenic carrier such as polylysine, they can become immunogenic and induce an immune response. These small molecules can bind to effector products and become antigenic, but are only immunoreactive and not immunogenic, and are also called incomplete antigens.

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

[0005] Small molecule antigens or haptens lack two or more sites for sandwich assays, so the double antibody sandwich assay cannot be used. Instead, a competitive assay is often used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen for binding to the solid-phase antibody. The greater the amount of antigen in the specimen, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This assay is often used for ELISA assays of small molecule hormones, drugs, etc.

[0006] Phenytoin (PTN), as a specific example of a hapten, is an exogenous small molecule anti-epileptic drug. It is not present in normal human serum or plasma, and its sodium salt form is generally used clinically.

[0007] As a traditional anti-epileptic drug, phenytoin sodium has a pharmacological mechanism of action that mainly blocks voltage-dependent Na + channel, reducing Na +The inward current stabilizes the neuronal cell membrane, reduces its excitability, and thus prevents local 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 6 to 10 days of continuous administration. Its low price and proven efficacy make it widely used clinically. However, due to its unique pharmacokinetics and narrow therapeutic window, it can easily lead to drug overdose and poisoning, and the diverse clinical manifestations of poisoning can easily lead to misdiagnosis. Therefore, during clinical diagnosis, it is necessary to continuously monitor the drug concentration in the blood of phenytoin.

[0009] Typically, a blood concentration of 10-20 μg / ml achieves optimal therapeutic effects without causing toxic effects. However, blood concentrations above 20 μg / ml can cause toxicity to the liver and kidneys, leading to coma and death. Generally, within 2-4 weeks of discontinuing medication, phenytoin blood concentrations can be reduced to 0.5 μg / ml or lower after metabolism by the liver and kidneys.

[0010] Currently known methods for detecting phenytoin include enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay (HEIA), chemiluminescence immunoassay (CLI), high-performance liquid chromatography (HPLC), and fluorescence polarization. However, these methods all have numerous drawbacks. For example, the radioimmunoassay (RIA) isotopes carry radioactive contamination, have a short shelf life, and are inconvenient to use. ELISA is also complex and time-consuming, making it unsuitable for clinical use. While CLI offers greater sensitivity, it requires specialized equipment and is expensive to use, hindering widespread adoption. Homogeneous enzyme immunoassay (EMIT) is the predominant method in clinical testing and diagnosis.

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

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

[0013] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided. Different from the 6-phosphate glucose dehydrogenase mutant disclosed in the previously published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphate glucose dehydrogenase mutant of the present application comprises mutations selected from the group consisting of: D306C, G426C, and D375C.

[0014] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided, wherein the 6-phosphate glucose dehydrogenase mutant is represented by a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4.

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

[0016] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

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

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

[0019] 43, 44, 45, 46, 47, 48, 49, 50.

[0020] In some specific embodiments, the molar ratio of the 6-phosphate glucose dehydrogenase mutant of the present application to the hapten is preferably 1:1.

[0021] In some specific embodiments, the molecular weight of the hapten is 100Da to 4000Da, 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, 1000, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210 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 the present application, the skilled artisan will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as phenytoin) that do not themselves carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker to facilitate covalent binding with a sulfhydryl group. Therefore, in the present application, a hapten derivative refers to a hapten that has been modified to carry a sulfhydryl reactive group.

[0023] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, 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 acid, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma galanin, 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, trans-triiodothyronine, free thyroxine, free triiodothyronine Amine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin II, 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, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0025] In specific embodiments, the hapten is phenytoin or a derivative thereof.

[0026] In a specific embodiment, the hapten is a phenytoin derivative having a sulfhydryl reactive group, such as imide, 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, which comprises the conjugate of the present application.

[0029] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a phenytoin detection reagent.

[0030] According to some embodiments, there is provided use of the conjugate of the present application in preparing a phenytoin detection reagent.

[0031] In a specific embodiment, the detection reagent is selected from: enzyme-linked immunosorbent assay detection reagent, chemiluminescence immunoassay detection reagent, homogeneous enzyme immunoassay detection reagent, latex-enhanced immunoturbidimetric detection reagent.

[0032] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

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

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

[0035] - a second reagent, which comprises the conjugate of the present 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 comprising 10 mM to 500 mM buffer, 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,

[0041] 5mM to 25mM substrate,

[0042] 0.1mg / L to 5mg / L phenytoin antibodies,

[0043] 10mM to 300mM NaCl,

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

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

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

[0047] A second reagent comprising:

[0048] 10mM to 500mM buffer,

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

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

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

[0052] 0.1g / L to 5g / L preservatives.

[0053] In some embodiments, the buffer is selected from one or a combination of the following: tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, tris(hydroxymethyl)methane buffer; preferably, phosphate buffer; the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 100 mM; the pH of the buffer 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, 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; and the azide is selected from sodium azide and lithium azide.

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

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

[0059] 1) providing a phenytoin derivative, preferably providing the phenytoin derivative in an aprotic solvent;

[0060] 2) providing the 6-phosphate glucose dehydrogenase mutant as defined in claim 2, preferably providing the 6-phosphate glucose dehydrogenase mutant in a buffer;

[0061] 3) contacting the phenytoin derivative and the 6-phosphate glucose dehydrogenase mutant at 18° C. to 28° C. for 1 to 4 hours, preferably 2 to 3 hours, to allow the phenytoin derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0062] 4) optionally, purifying the conjugate, preferably desalting;

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

[0064] The buffer is selected from the group consisting of PBS, Tris, TAPS, and TAPSO, and the pH of the buffer is between 6.0 and 8.0;

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

[0066] Preferably, before step 3), the 6-phosphate glucose dehydrogenase mutant contains a free thiol group; more preferably, the 6-phosphate glucose dehydrogenase mutant has a free thiol group at position 306, 375 or 426. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 .Structure diagram of phenytoin.

[0068] Figure 2 .Structure 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 .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0072] Figure 3D .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[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, and the mixture was heated to 50°C for 16 h.

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

[0078] 2. Synthesis of Compound 3

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

[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) was added dropwise. HATU (147 mg, 0.39 mmol) was added, and 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 product was purified by preparative chromatography to obtain a phenytoin derivative (100 mg, 67%).

[0082]

[0083] The purpose of this embodiment is to provide phenytoin with a group that can bind to the enzyme.

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

[0085] According to the G6PDH-phenytoin conjugate of the present application, the conjugation is carried out in the following manner: the sulfhydryl reactive group (such as but not limited to the maleimide group) on the phenytoin derivative molecule is covalently bound to the sulfhydryl 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 (mutant of the present application or mutant of the prior art), PB 100 mmol, NaCl 100 mmol, pH = 8.0;

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

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

[0091] 2. Coupling operation: 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 the reaction system was shaken at room temperature for 4 hours, it was eluted using a desalting column with the desalting solution, and the protein peak was collected. The obtained product was G6PDH-phenytoin conjugate.

[0093] Example 3. Preparation of kit

[0094] The following phenytoin detection kit was prepared, comprising:

[0095] Reagent R1, containing:

[0096] 100 mM PB buffer, pH 7.2

[0097] 15 mM glucose 6-phosphate

[0098] 15 mM β-nicotinamide adenine dinucleotide

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

[0100] 150mM NaCl

[0101] 1g / L bovine serum albumin

[0102] 1g / L Tween20

[0103] 1g / L sodium azide;

[0104] Reagent R2, including:

[0105] 100 mM PB buffer, pH 7.2

[0106] 0.1mg / L G6PDH-phenytoin conjugate

[0107] 1g / L bovine serum albumin

[0108] 1g / L Tween 20

[0109] 1g / L sodium azide;

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

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

[0112] Test example

[0113] Reaction time: 10 minutes, including a 4.7-minute incubation time. After adding reagent R2 and incubating for 1 minute, measure the absorbance A1. After another 1-minute incubation, measure the absorbance A2. 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 performances were total imprecision, repeatability, recovery, linearity, and 37°C accelerated stability.

[0115] Table 1. Parameters of fully automatic biochemical analyzer

[0116]

[0117]

[0118] Test Example 1. Absorbance calibration of phenytoin test kit

[0119] Table 2. Absorbance calibration of phenytoin detection kit

[0120]

[0121] Note: The mutant code-named A45C in the prior art has a mutation site corresponding to Figure 3A No. 46.

[0122] Test Example 2. Total Imprecision of Phenytoin Test Kit

[0123] Table 3. Total imprecision

[0124]

[0125] Test Example 3. Repeatability of Phenytoin Test Kit

[0126] Table 4. Repeatability

[0127]

[0128] Test Example 4. Phenytoin Test Kit Recovery

[0129] Table 5. Recycling

[0130]

[0131]

[0132] Test Example 5. Phenytoin Test Kit Linearity

[0133] Table 6. Linearity

[0134]

[0135] Test Example 6.37℃ Accelerated Stability

[0136] Table 7. Accelerated stability at 37°C

[0137]

[0138]

[0139] After the reagent of the present application is accelerated at 37°C for 7 days, the standardized absorbance decreases by about 15%, while the control reagent has a standardized absorbance of about 95% after being accelerated at 37°C for 7 days.

[0140] Test Example 7. Antibody Inhibition Rate

[0141] 1. Principle of Antibody Inhibition Rate Detection

[0142] When the antibody binds to the G6PDH-phenytoin conjugate, the G6PDH enzyme activity is affected due to steric hindrance, 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 is compared. This difference is reflected in the antibody's ability to inhibit G6PDH.

[0143] 2. Reaction system

[0144] Table 8. Preparation of antibody inhibition rate detection reagents

[0145]

[0146] Table 9. Antibody inhibition rate test parameters

[0147] Detection model Abbott C16000 Analysis / Time / Point Speed / 10min / 28-32 points R1 / S 120:20 Wavelength (secondary / primary) 405 / 340 Reaction type Increment

[0148] 3. Results

[0149] By comparing the absorbance values ​​of the G6PDH-phenytoin conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0150] Antibody inhibition rate = [1-(absorbance change value of G6PDH-phenytoin in the presence of antibody / absorbance change value of G6PDH-phenytoin in the absence of antibody)] × 100%.

[0151] Compared with previously published mutation sites, the mutants in this application have significantly improved the antibody inhibition rate, reaching over 30% and up to 55%, while the inhibition rate of previously commonly used mutation sites (such as A45C and K55C) is only around 40% or even lower.

[0152] Table 10. Antibody inhibition rates of different G6PDH mutants

[0153]

[0154] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants in the prior art, the mutation site (i.e., the site where the free thiol group is introduced) in the enzyme mutants of the present application (D306C, D375C, G426C) is the location where coupling occurs with the hapten (such as a hormone, a small molecule drug, etc.). When the hapten binds to the hapten-specific antibody at this position, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme. At the same time, after the mutation is introduced, it cannot substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is very important, and it is necessary to take into account the activity of the G6PDH enzyme, the spatial folding of the coupled molecule, and the full exposure of the hapten epitope.

[0155] Since the mutant of the enzyme has a significant improvement in the antibody inhibition rate, after the conjugate of the mutant of the enzyme and phenytoin is prepared into a kit, the reagent has a significant performance improvement in repeatability, total imprecision, linearity, stability and other properties.

Claims

1. Use of the conjugate in the preparation of phenytoin detection reagents: The conjugate is formed by coupling a 6-phosphate glucose dehydrogenase mutant with a phenytoin derivative in a molar ratio of 1:1; The phenytoin derivative is shown in the following formula I: Formula I; Compared to wild-type 6-phosphate glucose dehydrogenase, the 6-phosphate glucose dehydrogenase mutant comprises a mutation D306C; The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No.

2.

2. The method according to claim 1, wherein the detection reagent is a homogeneous enzyme immunoassay detection reagent.

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