Digoxin detection kit

Abstract

This application relates to a digoxin detection kit. Specifically, the 6-glucose phosphate dehydrogenase mutant of this application comprises one or a combination of the following mutations compared to wild-type 6-glucose phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the 6-glucose phosphate dehydrogenase mutant of this 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 patent application 202010004879.2 "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Digoxin Detection Reagent" filed on January 3, 2020. Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a mutant enzyme glucose-6-phosphate dehydrogenase (G6PDH for short) and its application in a digoxin 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] The structural formula of digoxin is shown below:

[0007]

[0008] Digoxin is a cardiac glycoside that has a positive inotropic effect on the heart, slowing the heart rate and inhibiting cardiac conduction. It is used to treat acute and chronic heart failure, including hypertension, valvular heart disease, and congenital heart disease. It is particularly suitable for heart failure accompanied by atrial fibrillation with a rapid ventricular rate.

[0009] Common adverse reactions include proarrhythmic effects, nausea, vomiting, lower abdominal pain, and abnormal weakness. Less common reactions include blurred vision and central nervous system reactions (such as depression or confusion). Rare reactions include drowsiness, headache, rash, and urticaria (allergic reaction). Among the manifestations of digitalis poisoning, proarrhythmia is the most important, followed by atrioventricular block, paroxysmal or accelerated junctional tachycardia, paroxysmal atrial tachycardia with atrioventricular block, ventricular tachycardia, sinus arrest, and ventricular fibrillation.

[0010] Therefore, attention should be paid to monitoring the adverse reactions of this drug. Moreover, due to individual differences in drug metabolism, blood drug concentration monitoring should be combined with clinical use to formulate a reasonable dosing regimen and minimize the occurrence of adverse reactions.

[0011] Currently known methods for detecting digoxin include high-performance liquid chromatography (HPLC), chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, and latex agglutination turbidimetry. HPLC requires complex sample pretreatment, is complex and time-consuming, and is expensive. Luminescence immunoassay reagents are expensive, making them unsuitable for routine therapeutic drug testing and hindering widespread adoption. Existing homogeneous enzyme immunoassays and latex agglutination turbidimetry are often limited in their application due to complex preparation processes and large batch-to-batch variability.

[0012] Prior art CN108593905A describes a digoxin detection kit and its preparation method. However, this prior art method relies on activating the reactive groups of the small molecule drug (digoxin) itself before reacting with the enzyme. This coupling method can result in multiple digoxins being linked to the same glucose-6-phosphate dehydrogenase. Furthermore, it is difficult to ensure consistency in the coupling sites, making it difficult to ensure a directional 1:1 reaction between the small molecule drug and the enzyme, resulting in large batch variability. Summary of the Invention

[0013] In view of the needs in the field, the present application provides a novel 6-phosphate glucose dehydrogenase mutant and its use in preparing a digoxin detection kit.

[0014] 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, D375C, and G426C.

[0015] 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.

[0016] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0017] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0018] 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).

[0019] 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.

[0020] 43, 44, 45, 46, 47, 48, 49, 50.

[0021] 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.

[0022] 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.

[0023] 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 digoxin) 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.

[0024] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0025] Haptens include, but are not limited to, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin and digitoxin), 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, 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.

[0026] In specific embodiments, the hapten is digoxin or a derivative thereof.

[0027] In a specific embodiment, the hapten is a digoxin derivative that carries a sulfhydryl-reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0028] In a specific embodiment, the hapten is a digoxin derivative, as shown in Formula I:

[0029]

[0030] in,

[0031]

[0032] In some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

[0033] In some specific embodiments, the digoxin derivative has the structure shown in Formula I-1:

[0034]

[0035] in,

[0036]

[0037] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0038] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a digoxin detection reagent.

[0039] According to some embodiments, there is provided use of the conjugate of the present application in preparing a digoxin detection reagent.

[0040] 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.

[0041] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0042] According to some embodiments, there is provided use of the conjugate of the present application in preparing a digoxin detection device.

[0043] In a specific embodiment, the detection device can be prepared in the form of a well plate (eg, a 96-well plate), for example, the plate is coated with the reagent according to the present application.

[0044] In a specific embodiment, the detection device can be prepared in the form of particles (such as latex, magnetic beads), for example, the particles are coated with the reagent according to the present application.

[0045] According to some embodiments, a digoxin detection kit is provided, comprising:

[0046] - a first reagent, comprising a substrate, a buffer, and a digoxigenin antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0047] - a second reagent, comprising the conjugate of the present application and a buffer;

[0048] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 ng / ml to 5 ng / ml digoxin (e.g. 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 ng / ml or any value in between); and

[0049] - Optionally, a control comprising 10 mM to 500 mM buffer, 0.7 ng / ml to 4.5 ng / ml (e.g. 0.7, 0.8, 1.5, 1.8, 2, 3, 4, 4.5 ng / ml or any value in between) digoxin.

[0050] According to one embodiment, a digoxin detection kit is provided, comprising:

[0051] The first reagent comprises:

[0052] 10mM to 500mM buffer,

[0053] 5mM to 50mM substrate,

[0054] 0.01μg / ml to 10μg / ml digoxin antibody (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, 2, 3, 4, 5μg / ml),

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

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

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

[0058] A second reagent comprising:

[0059] 10mM to 500mM buffer,

[0060] 0.01 μg / ml to 10 μg / ml of the conjugate according to the present application (0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 μg / ml),

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

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

[0063] 0.1g / L to 5g / L preservatives.

[0064] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, 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 50 to 100 mM; the pH of the buffer is 7 to 8.

[0065] 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.

[0066] In some embodiments, the surfactant is selected from one or a combination of the following: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0067] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, biopreservative PC (such as PC-300), thimerosal; the azide is selected from sodium azide, lithium azide, PC-300.

[0068] In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

[0069] In some specific embodiments, the digoxin antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, or human.

[0070] In some specific embodiments, the digoxin antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment.

[0071] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0072] 1) providing a digoxin derivative according to the present application, in particular providing a digoxin derivative according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0073] 2) providing a 6-phosphate glucose dehydrogenase mutant, preferably providing the 6-phosphate glucose dehydrogenase mutant in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, wherein the buffer has a pH of 6.0 to 8.0);

[0074] 3) contacting the 6-phosphate glucose dehydrogenase mutant and the digoxin derivative at a molar ratio of digoxin derivative: enzyme = 500:1 to 1:500 (preferably 50:1 to 1:50) at 18° C. to 28° C. for 1 to 4 hours (1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or any value therebetween, preferably 2 to 3 hours) to allow the digoxin derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0075] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0076] In some embodiments, the contact molar ratio of the hapten to the enzyme in the reaction system is 1:n, wherein n is 1 to 500, 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, 100, 200, 300, 400, 500, and ranges between any of the above values.

[0077] In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0078] In some specific embodiments, prior to conjugation, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing for a directed reaction with digoxin.

[0079] Wild-type 6-phosphate glucose dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific embodiments, 6-phosphate glucose dehydrogenase is genetically engineered so that the amino acid at a specific site (306, 375 or 426) is mutated to cysteine, thereby carrying a free sulfhydryl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0081] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0082] Figure 3.G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0083] Figure 4 .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0084] Example

[0085] Example 1. Synthesis of digoxin derivatives

[0086]

[0087] 1. Synthesis of Compound 2

[0088] 1.0 g of digoxin was dissolved in 95% ethanol (80 ml), and then a solution of periodic acid (1.0 g) in water (10 ml) was added. The mixture was stirred at room temperature (18-28° C.) for 1 hour. The residue was removed by filtration, the solvent was removed under reduced pressure, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to obtain compound 2 (white solid, 0.99 g, 99%).

[0089] 2. Synthesis of Compound 4

[0090] Compound 2 (900 mg, 1.16 mmol) was dissolved in 10 ml of dry methanol, and compound 3 (318 mg, 1.0 mmol) was added to the reaction system. The mixture was stirred at room temperature for 5 minutes. Sodium cyanoborohydride (146 mg, 2.32 mmol) was added, and the mixture was stirred at room temperature for approximately 12 hours. The solvent was removed under reduced pressure, and the mixture was directly purified by column chromatography to obtain compound 4 (white solid, 585 mg, 66%).

[0091] 3. Synthesis of Compound 5

[0092] Compound 4 was dissolved in 15 ml of dichloromethane and stirred at room temperature for 30 minutes under nitrogen atmosphere, then 10 ml of piperidine was added and stirred at room temperature for 2 hours. The solvent was removed under reduced pressure and purified by column chromatography to obtain compound 5 (460 mg, 78%).

[0093] 4. Synthesis of digoxin derivatives

[0094] Compound 5 (88 mg, 0.11 mmol) and compound 6 (17 mg, 0.11 mmol) were dissolved in DCM (8 mL), triethylamine (33 mg, 0.33 mmol) was added dropwise, and then HATU (50 mg, 0.13 mmol) was added. The mixture was stirred at room temperature for 2 h to give a digoxin derivative (white solid, 50 mg, 45%).

[0095] 5. The structure of digoxin derivatives was confirmed to be correct by mass spectrometry and nuclear magnetic resonance.

[0096] In this embodiment, digoxin is provided with a group that can bind to an enzyme.

[0097] Example 2. Coupling of digoxin derivatives with G6PDH molecules

[0098] 1. Coupling method of the present application

[0099] According to the G6PDH-digoxin 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 digoxin derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0100] 1. Dissolve the digoxin derivative prepared in Example 1 in N,N-dimethylformamide (10 mg / ml);

[0101] 2. G6PDH solution: G6PDH (e.g., the mutant of the present application) was dissolved in 100 mmol PB, 100 mmol NaCl, pH = 8.0;

[0102] 3. Add 200 μl of G6PDH solution to 750 μl of buffer solution (0.05 M Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH = 7.2); then add 50 μl of digoxin derivative N,N-dimethylformamide solution;

[0103] 4. The mixed solution was shaken thoroughly at room temperature (18-28°C) for 2-3 hours, desalted, and the protein peak was collected. The obtained product was G6PDH-digoxigenin conjugate.

[0104] 2. Control coupling method (refer to the method of CN108593905A)

[0105] Accurately weigh 100-300 mg of digoxin and dissolve it in 5-15 mL of anhydrous ethanol;

[0106] Add 5-15 mL of 10-200 mM sodium periodate dropwise to the above solution, shake gently, and stir at room temperature for 0.5-2 hours;

[0107] Add 0.5-1 mL of 0.5-2 M ethylene glycol dropwise and stir at room temperature for 5-10 minutes;

[0108] Add the above reaction mixture dropwise to 5-15 mL of a stirring 2-3% G6PDH solution, and adjust the solution pH to 9.0-9.5. Continue stirring and reacting for 0.5-2 hours to stabilize the solution pH.

[0109] Add 100-200 mg of sodium tetrahydroborate and stir to reduce for 12-24 hours;

[0110] The G6PDH-digoxigenin conjugate was purified by G-25 gel chromatography column.

[0111] Example 3. Preparation of kit

[0112] Prepare the following kit for detecting digoxin, which contains:

[0113] Reagent R1, containing:

[0114] TAPS buffer 100 mM, pH 7.0

[0115] 15 mM glucose 6-phosphate

[0116] 15 mM β-nicotinamide adenine dinucleotide

[0117] 0.5 μg / ml digoxin antibody (commercially available antibody, no special restrictions)

[0118] 1g / L bovine serum albumin

[0119] 1g / L Brij

[0120] 1g / L sodium azide;

[0121] Reagent R2, including:

[0122] Phosphate buffer 200 mM, pH 8.0

[0123] 0.1 μg / ml G6PDH-digoxigenin conjugate

[0124] 100mM NaCl

[0125] 1g / L bovine serum albumin

[0126] 1g / L Brij

[0127] 1g / L sodium azide;

[0128] Calibrators: 20 mM HEPES buffer, and 0 ng / ml, 0.5 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, 5 ng / ml digoxin (or as needed);

[0129] Quality control: 20 mM HEPES buffer, and 0.6-0.8 ng / ml, 1.6-2.0 ng / ml, 3.8-4.2 ng / ml digoxin (or add as needed).

[0130] The above reagents (optionally including quality control products and calibrators) are assembled into a digoxin homogeneous enzyme immunoassay kit.

[0131] Test example

[0132] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, the enzyme-labeled antigen (such as G6PDH-digoxin) competes with the unlabeled antigen (digoxin) for binding with a quantitative antibody (digoxin antibody). 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.

[0133] By detecting the absorbance change of NADH at a wavelength of 340nm, the digoxin content in the liquid can be calculated.

[0134] Table 1. Parameters of fully automatic biochemical analyzer

[0135]

[0136]

[0137] Test Example 1. Performance of the kit of this application

[0138] 1. Calibration Experiment

[0139] Table 2. Absorbance of the calibration of the digoxin detection kit

[0140]

[0141] 2. Precision experiment

[0142] Table 3. Total imprecision

[0143]

[0144] 3. Repeatability

[0145] Table 4. Repeatability

[0146]

[0147]

[0148] 4. Recycling test

[0149] Table 5. Recovery data

[0150]

[0151] 5. Linearity Experiment

[0152] Table 6. Linearity

[0153]

[0154]

[0155] Test Example 2: Airborne Stability

[0156] After the reagent of the present application (D375C mutant) was accelerated at 37°C for 7 days, the absorbance of the calibration decreased by less than 10%, while after the control reagent was accelerated at 37°C for 7 days, the absorbance of the calibration decreased significantly.

[0157] Table 7. Accelerated stability at 37°C

[0158]

[0159] Test Example 3. Antibody Inhibition Rate

[0160] 1. Principle of Antibody Inhibition Rate Detection

[0161] When the antibody binds to the G6PDH-digoxin 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.

[0162] 2. Reaction system

[0163] Table 8. Preparation of antibody inhibition rate detection reagents

[0164]

[0165] 3. Results

[0166] By comparing the absorbance values ​​of the G6PDH-digoxin conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0167] Antibody inhibition rate = absorbance change of G6PDH-digoxin conjugate in the presence of antibody / absorbance change of G6PDH-digoxin in the absence of antibody × 100%.

[0168] Compared to the previously published mutation site (A45C), the mutants described in this application showed significantly improved enzyme activity retention, reaching over 40% (G426C: 40%; D375C: 49%), with a maximum of 62% (D306C). When the previously published mutation sites (e.g., A45C and K55C) were prepared into G6PDH-digoxin conjugates according to the methods described in this application, the inhibition rates were only 33% and 40%.

[0169] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants (A45C, K55C) in the prior art, the mutation site in the enzyme mutant of the present application (i.e., the site where the free thiol group is introduced) 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.

[0170] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the reagent kit, after the enzyme mutant is coupled with digoxin, has a significant improvement in performance in terms of inter-batch coefficient of variation, linearity, repeatability, stability, etc.

Claims

1. A digoxin detection kit comprising: A first reagent comprising a substrate, a digoxigenin antibody, and a buffer; a second reagent comprising a conjugate and a buffer; The conjugate is formed by coupling a 6-phosphate glucose dehydrogenase mutant with a digoxin derivative in a molar ratio of 1:1; The digoxin derivative is represented by formula I-1: in, Compared to wild-type 6-phosphate glucose dehydrogenase, the 6-phosphate glucose dehydrogenase mutant comprises a D306C mutation; The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No.

2.

2. The digoxin detection kit according to claim 1, further comprising a calibrator and a quality control product; The calibrator contains 10mM to 500mM buffer and 0ng / ml to 5ng / ml digoxin; The quality control contains 10 mM to 500 mM buffer and 0.7 ng / ml to 4.5 ng / ml digoxin.

3. The digoxin detection kit according to claim 1, comprising: The first reagent comprises: 10mM to 500mM buffer, 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 0.01μg / ml to 10μg / ml digoxin antibody, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, 0.1g / L to 5g / L preservatives; A second reagent comprising: 10mM to 500mM buffer, 0.01 μg / ml to 10 μg / ml of the conjugate, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, 0.1g / L to 5g / L preservatives; The buffer is selected from the group consisting of: TAPS buffer, phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer, and HEPES buffer; The pH of the buffer is 7 to 8; The stabilizer is selected from the group consisting of bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; The surfactant is selected from the group consisting of: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, and AEO7; The preservative is selected from the group consisting of: azide, MIT, biological preservative PC, and thimerosal.

4. The digoxin detection kit according to claim 3, comprising: The first reagent comprises: 50mM to 300mM buffer, 10mM to 20mM glucose-6-phosphate, 10mM to 20mM oxidized β-nicotinamide adenine dinucleotide, 0.1μg / ml to 1μg / ml digoxin antibody, 1g / L to 5g / L stabilizer, 1g / L to 5g / L surfactant, 1g / L to 5g / L preservatives; A second reagent comprising: 50mM to 300mM buffer, 0.05 μg / ml to 0.5 μg / ml of the conjugate, 1g / L to 5g / L stabilizer, 1g / L to 5g / L surfactant, 1g / L to 5g / L preservatives.

5. The digoxin detection kit according to claim 3, wherein the preservative is selected from the group consisting of sodium azide, lithium azide, and PC-300.

6. The digoxin detection kit according to claim 3, comprising: The first reagent comprises: 50 mM TAPS buffer, pH 8.0, 15mM glucose-6-phosphate, 15mM oxidized β-nicotinamide adenine dinucleotide, 0.5μg / ml digoxin antibody, 1g / L bovine serum albumin, 1g / L Tween20, 1g / L sodium azide; A second reagent comprising: 50 mM Tris buffer, pH 8.0, 0.1 μg / ml of the conjugate, 1g / L bovine serum albumin, 1g / L Tween20, 1g / L sodium azide.

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