Use of conjugates in the preparation of detection reagents

Abstract

This application relates to the use of conjugates in the preparation of diagnostic reagents. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Diagnostic kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making them suitable for high-throughput detection.

Description

[0001] This application is a divisional application of patent application 201911372535.0, filed on December 27, 2019, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of digitalisin detection reagent”. Technical Field

[0002] This application relates to the field of biological detection, and in particular to a mutant enzyme, glucose-6-phosphate dehydrogenase (G6PDH), and its application in a digitalis glycoside 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 mode is often used. 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 assay of small molecule hormones and drugs.

[0006] The structural formula of digitoxin (DG) is shown below:

[0007]

[0008] Digitalisin belongs to a class of naturally occurring cardiac glycosides (CG), obtained from digitalis violaceum, digitalis pubescens, or other suitable digitalis.

[0009] Digitalisin is rapidly and completely absorbed in the gastrointestinal tract, but its metabolism in the body is slow, primarily in the liver, with most metabolites being inactive. Digoxin is commonly used for heart failure, specifically for congestive heart failure and arrhythmias. Due to its slow and prolonged action, it is particularly suitable for long-term use in patients with chronic heart failure. Digitalisin and Na... + / K + -ATPase reversibly binds to the cell membrane, preventing the enzyme from binding to ATP and inhibiting Na+. + With K + Active transport of Na+ within cells + Increase, K + The reduction is due to the direct electrophysiological effects and toxicity of digitalis glycosides.

[0010] The dosage of digitoxin should be carefully adjusted according to the individual patient's needs. The therapeutic steady-state plasma concentration of digitoxin is 10 to 25 ng / mL, and higher concentrations (30 ng / mL) may be accompanied by toxicity. Therefore, effective monitoring of digitoxin concentrations in patients is necessary in clinical practice.

[0011] Currently known methods for detecting digitoxin include chemiluminescence immunoassay, high-performance liquid chromatography (HPLC), gas-liquid chromatography (GC), gas chromatography, and mass spectrometry (MS). However, these methods all have several drawbacks. For example, while chemiluminescence has good sensitivity, it requires specialized equipment, resulting in high costs and hindering widespread adoption. In clinical diagnostic testing, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetric assays are the primary methods of detection.

[0012] The principle of homogeneous enzyme immunoassay: In a homogeneous liquid reaction system, enzyme-labeled antigen (such as G6PDH-digitoxin) and unlabeled antigen (digitoxin) compete for binding with a quantitative amount of antibody (digitoxin antibody). The more the antibody binds to the unlabeled antigen, the more activity is released from the enzyme-labeled antigen, 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 digitoxin in the liquid can be calculated.

[0013] Existing homogeneous enzyme immunoassays and latex agglutination turbidimetric assays are often limited in application due to complex preparation processes and large batch-to-batch variations. CN102768284A describes a method for preparing a small molecule drug-G6PDH enzyme conjugate. However, existing methods rely on activating the reactive groups of the small molecule drug itself before reacting with the enzyme. This strategy makes it difficult to ensure a directed reaction between the small molecule drug and the enzyme, leading to large batch-to-batch variations. Summary of the Invention

[0014] 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 digitalisin detection kit.

[0015] 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.

[0016] 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.

[0017] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0018] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0019] 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).

[0020] 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.

[0021] 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.

[0022] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 1:1.

[0023] 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.

[0024] 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., digitalisin) can be modified to have a linker for covalent binding with a thiol group. Therefore, in this application, hapten derivatives refer to haptens modified to possess a thiol-reactive group.

[0025] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0026] Haptens include, but are not limited to: theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin), 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 glycoproline, 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, and so on. The following substances are present in the blood: triiodothyronine, 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 peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, and osteocalcin.

[0027] In the specific implementation plan, the hapten is digitoxin or its derivative.

[0028] In a specific implementation, the hapten is a digitalis glycoside derivative with a thiol reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0029] In the specific implementation scheme, the hapten is a digitalisin derivative, as shown in Formula I:

[0030]

[0031] in,

[0032] In some implementations, m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

[0033] In some implementations, X is maleimide, bromoacetyl, vinyl sulfone, or aziridine.

[0034] Those skilled in the art will understand that the function of X is to react with the thiol group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine, and thiol groups is expected. Although specific groups are used in the examples, they are not intended to be limited thereto.

[0035] In some specific embodiments, the digitoxin derivative has a structure selected from the following formula:

[0036]

[0037]

[0038] m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6.

[0039] In some specific embodiments, the digitoxin derivative has a structure selected from the following formula:

[0040]

[0041] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0042] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of digitalisin detection reagents is provided.

[0043] According to some implementation schemes, the use of the conjugates of this application in the preparation of digitoxin detection reagents is provided.

[0044] 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.

[0045] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0046] According to some implementation schemes, a digitoxin detection kit is provided, comprising:

[0047] - First reagent, comprising a substrate, a buffer solution, and a digitalisin antibody; the substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0048] - A second reagent, comprising the conjugate and buffer solution of this application;

[0049] -Optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 ng / ml to 80 ng / ml digitoxin; and

[0050] -Optionally, a quality control product comprising 10 mM to 500 mM buffer solution and 10 ng / ml to 50 ng / ml digitoxin.

[0051] According to one embodiment, a digitoxin detection kit is provided, comprising:

[0052] The first reagent comprises:

[0053] 10mM to 500mM buffer solution

[0054] 5mM to 50mM substrate,

[0055] 10 ng / ml to 10 μg / ml of digitoxin antibody,

[0056] 0.1 g / L to 5 g / L stabilizer

[0057] 0.1 g / L to 5 g / L surfactant,

[0058] 0.1 g / L to 5 g / L of preservatives;

[0059] The second reagent comprises:

[0060] 10mM to 500mM buffer solution

[0061] 0.01 μg / ml to 10 μg / ml according to the conjugate of this application,

[0062] 0.1 g / L to 5 g / L stabilizer

[0063] 0.1 g / L to 5 g / L surfactant,

[0064] Preservatives ranging from 0.1 g / L to 5 g / L.

[0065] 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 from 7 to 8.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] In some embodiments, the substrate comprises: 6-phosphoglucose and β-nicotinamide adenine dinucleotide.

[0070] In some specific implementation schemes, the digitalisin antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0071] In some specific implementation schemes, the digitalisin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0072] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0073] 1) Provide digitalisin derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0074] 2) Provide a glucose-6-phosphate dehydrogenase mutant, preferably provided in a buffer (which provides a reaction environment, such as, but not limited to, PBS, Tris, TAPS, TAPSO, said buffer pH 6.0 to 8.0);

[0075] 3) At 18°C ​​to 28°C, the glucose-6-phosphate dehydrogenase mutant and the digitalisin derivative are contacted at a molar ratio of 1:n for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the digitalisin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

[0076] 4) The conjugate may be purified as needed, for example, by desalting.

[0077] 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.

[0078] In some specific implementations, steps 1) and 2) are interchangeable.

[0079] In some specific implementations, prior to coupling, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing a directed reaction with digitoxin.

[0080] Wild-type glucose-6-phosphate dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific implementations, glucose-6-phosphate dehydrogenase is genetically engineered to mutate the amino acid at a specific site (positions 306, 375, or 426) to cysteine, thereby giving it a free sulfhydryl group. Attached Figure Description

[0081] Figure 1 Structure diagram of digitalisin.

[0082] Figure 2 Structural diagram of digitalisin derivatives.

[0083] Figure 3A .G6PDH (wild type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0084] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0085] Figure 3C The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0086] Figure 3D The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0087] Example

[0088] Example 1. Synthesis of digitalisin derivatives

[0089]

[0090] 1. Synthesis of Compound 2

[0091] Dissolve 1.0 g of digitoxin in 80 ml of 95% ethanol, then add 10 ml of periodic acid aqueous solution (1.0 g of periodic acid) and stir at room temperature (18 to 28°C, preferably 20 to 25°C) for 1 hour.

[0092] 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 give compound 2 (white solid, 0.90 g, 90% yield).

[0093] 2. Synthesis of Compound 4

[0094] Compound 2 (900 mg, 1.18 mmol) was dissolved in 10 mL of dry methanol. Compound 3 (318 mg, 1.0 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 minutes. Sodium cyanoborohydride (146 mg, 2.32 mmol) was then added, and the mixture was stirred at room temperature for approximately 12 hours.

[0095] The solvent was removed under reduced pressure, and the compound was purified by direct column chromatography to give compound 4 (white solid, 400 mg, 46%).

[0096] 3. Synthesis of Compound 5

[0097] 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 the mixture was stirred at room temperature for 2 hours. The solution was removed under reduced pressure, and compound 5 (500 mg, 85%) was purified by column chromatography.

[0098] 4. Synthesis of digitalisin derivatives

[0099] Compound 5 (88 mg, 0.11 mmol) and compound 6 (17 mg, 0.11 mmol) were dissolved in 8 mL of DCM, and triethylamine (33 mg, 0.33 mmol) was added dropwise, followed by HATU (50 mg, 0.13 mmol). The mixture was stirred at room temperature for 2 h to obtain the digitalisin derivative (white solid, 48 mg, 43%).

[0100] The product structure was confirmed using conventional methods. In this embodiment, digitoxin contains a group that can bind to an enzyme.

[0101] Example 2. Coupling of digitalisin derivatives with G6PDH molecules

[0102] According to the G6PDH-digitoxin 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 digitoxin derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0103] 1. Solution preparation:

[0104] Digitalisin derivative solution: 10 mg / ml of the digitalisin derivative prepared in Example 1 was dissolved in DMF;

[0105] G6PDH solution: G6PDH (the mutant of this application or the prior art mutant) is dissolved in 100 mmol PB, 100 mmol NaCl, pH=8.0;

[0106] Coupling solution: 100mM PB / K, 100mM EDTA, 150mM NaCl, pH=7.2;

[0107] Desalination solution: 100mM PB / K, 0.1% NaN3, 1% NaCl, pH=8.0.

[0108] 2. Coupling operation:

[0109] 2 ml of G6PDH solution, 7.5 ml of coupling solution and 0.5 ml of digitalisin derivative solution were reacted at room temperature for 4 h.

[0110] 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-digitoxin conjugate.

[0111] Example 3. Preparation of the reagent kit

[0112] Prepare the following kit for the detection of digitoxin, comprising:

[0113] Reagent R1 contains:

[0114] 50mM HEPES, pH 7.0

[0115] 10mM glucose-6-phosphate

[0116] 10mM β-nicotinamide adenine dinucleotide

[0117] 50 ng / ml digitoxin antibody (commercially available antibody, no special restrictions)

[0118] 1 g / L bovine serum albumin

[0119] 1g / L Tween20

[0120] 1 g / L sodium azide;

[0121] Reagent R2 includes:

[0122] 200mM Tris buffer, pH 8.0

[0123] 0.1 μg / ml G6PDH-digitoxin conjugate

[0124] 1 g / L bovine serum albumin

[0125] 1g / L Tween 20

[0126] 1 g / L sodium azide;

[0127] Calibrator: 20 mM HEPES buffer, and 0.0, 5.0, 10.0, 20.0, 40.0, 80.0 ng / ml digitoxin (or add as needed);

[0128] Quality control: 20mM HEPES buffer, and 8ng / ml, 15ng / ml, and 35ng / ml digitoxin (or add as needed).

[0129] Assemble the above reagents (optionally including quality control and calibrators) into a homogeneous enzyme immunoassay kit for digitalisin.

[0130] Detection example

[0131] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0132] model Hitachi 7180 Analysis points [Rate-A]

[10]

[25]

[34] Wavelength (SUB / MAIN)

[410] /

[340] S.VIL

[10] S.R1; S.R3

[150] ；

[50] ABS.LIMIT

[32000] [Incrementing] Calibration type [Spline] POINT [6]SPAN PONIT[6] Calibrator 0.0, 5.0, 10.0, 20.0, 40.0, 80.0ng / ml sample The samples to be tested are various physiological samples, such as serum and plasma.

[0133] Test Example 1. Accuracy, precision, and linearity experiments of the kit in this application (D306C mutant)

[0134] Table 2. Accuracy and Precision

[0135]

[0136] Table 3. Linear

[0137] Test 1 Test 2 Test 3 mean Theoretical value relative deviation absolute deviation 1 1.81 2.08 1.68 1.86 1.28 - 0.58 2 7.30 7.19 7.66 7.38 7.38 0.1％ 0.01 3 13.22 12.90 12.83 12.98 13.48 -3.7％ -0.49 4 20.16 20.45 19.59 20.07 19.58 2.5％ 0.49 5 24.39 25.65 24.89 24.98 25.68 -2.7％ -0.70 6 32.00 30.43 31.24 31.22 31.78 -1.7％ -0.55 7 38.84 39.04 39.20 39.03 37.87 3.0％ 1.15 8 44.62 42.71 44.01 43.78 43.97 -0.4％ -0.19 9 48.80 51.18 50.15 50.04 50.07 -0.1％ -0.03 10 57.52 54.13 54.70 55.45 56.17 -1.3％ -0.72 11 62.24 61.35 61.54 61.71 62.27 -0.9％ -0.56 12 67.93 68.98 69.58 68.83 68.37 0.7％ 0.46 13 74.25 78.44 72.43 75.04 74.47 0.8％ 0.57

[0138] Example 2. Recovery Experiment

[0139] Four concentrations of digoxin (USP) were prepared by adding it to the mixed serum samples, and each level was measured 21 times. The mean and bias were calculated.

[0140] Table 4. Recycling Experiment

[0141]

[0142]

[0143] Example 3. Common Drug Interference Prevention

[0144] Table 5. Results of anti-interference measurement

[0145] serial number Interference Concentration (μg / ml) 1 N-acetylcysteine 150 2 Amitriptyline 2 3 Ampicillin sodium 100 4 K-hydroxybenzenesulfonate (potassium hydroquinone sulfonate) 200 5 Hydroxyzine dihydrochloride 1 6 Methyldopa sesquihydrate 20 7 Promethazine 100 8 ascorbic acid 30 9 tetracycline 50 10 Acetylsalicylic acid 1000 11 Probenecid 500 12 Levodopa (3,4-dihydroxy-L-phenylalanine) 20 13 Metronidazole 100 14 phenothiazine 200 15 Ibuprofen 500 16 phenylbutazone 16 17 Acetaminophen 200 18 Chlorpromazine 100 19 Kapokaloids 50 20 nortriptyline 1 21 Cefoxitin 2500 22 Cetirizine dihydrochloride 3 23 Cyclosporine 5 24 Dishipamin 3 25 Ethylbenzene 50 26 5-(p-hydroxyphenyl-5-phenylhydrazine) 1000 27 Imipramine 6 28 Phenobarbital 500 29 Phenytoin sodium 500 30 Theophylline 100 31 valproic acid 1000 32 Ethosuccinylamine 1000 33 Primafenone 1000 34 Rifampicin 60

[0146] Detection Example 4. Cross-reactivity

[0147] Digoxin was dissolved in buffer solution to prepare digitoxin calibrators at different concentrations. The following concentrations of digitoxin analogues were then added to the mixed serum samples.

[0148] The reagents used in this application were calibrated using digitalisin calibrators. The mixed serum sample and the above sample were measured 5 times respectively. The ratio of the measured value of the above sample to the measured value of the mixed serum sample is the cross-reactivity rate.

[0149] Table 6. Cross-reactivity rate

[0150] Chemical name This application reagents 5.00 ng / ml digoxin glycoside 309.7％ 20 ng / mL isohydroxydigitoxin 2.43％ 20 ng / mL digoxigenin-Mono 0.08％ 20 ng / mL digoxigenin-bis 1.60％ 20 ng / mL digoxin 1.05％ 750ug / mL strophanthin 1.85％ 750ug / mL deslanoside 0.33％

[0151] Example 5. Inter-batch variation of the digitoxin detection kit

[0152] Three batches of the reagents used in this application (including the D306C mutant) were calibrated separately, and the differences in absorbance changes among the different batches were calculated.

[0153] Table 7. Calibration data between batches

[0154]

[0155] Table 8. Comparison between batches

[0156]

[0157] The control kit (which differs from the kit of this application in that the enzyme is replaced with the A45C mutant in the prior art) was used to determine the batch-to-batch difference using the same method, and the CV was in the range of 2.9% to 4.5%, which was significantly lower than that of the kit of this application.

[0158] Example 6. Antibody inhibition rate

[0159] 1. Detection principle of antibody inhibition rate

[0160] When the antibody binds to the G6PDH-digitoxin 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.

[0161] 2. Reaction system:

[0162] Table 9. Preparation of reagents for detecting antibody inhibition rate

[0163]

[0164] 3. Results:

[0165] By comparing the absorbance values ​​of the G6PDH-digitoxin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0166] Antibody inhibition rate = [1 - (change in absorbance of G6PDH-digitoxin with antibody / change in absorbance of G6PDH-digitoxin without antibody)] × 100%.

[0167] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in antibody inhibition rate, reaching over 32% (G426C: 32%; D375C: 47%), with a maximum of 55% (D306C). Previously published mutation sites (e.g., A45C, K55C) have inhibition rates ranging from 30% to 44%.

[0168] While not limited to specific theories, it can be partially explained as follows:

[0169] Compared to existing G6PDH mutants (A45C, K55C), the mutation sites (i.e., the sites introducing free sulfhydryl groups) in the enzyme mutants of this application (especially D306C, D375C) 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.

[0170] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the formulation of a kit by conjugating the enzyme mutant with digitoxin shows a significant improvement in performance in terms of batch-to-batch coefficient of variation, linearity, and specificity.

Claims

1. Uses of conjugates in the preparation of detection reagents: The detection reagent is a homogeneous enzyme immunoassay reagent for the detection of digitoxin; The conjugate is formed by conjugating a glucose-6-phosphate dehydrogenase mutant with a digitalisin derivative. The digitalisin derivative is selected from the structure shown in the following formula: in, Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains the D306C mutation; the glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No. 2.

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