Methods of preparing conjugates

Abstract

The present application relates to a preparation method of a conjugate. Specifically, the 6-phosphogluconate dehydrogenase mutant of the present application comprises one mutation or a combination thereof selected from D306C, D375C, G426C compared with wild-type 6-phosphogluconate dehydrogenase. The detection kit prepared by using the 6-phosphogluconate dehydrogenase mutant of the present application has high specificity, high sensitivity, convenient operation, short detection time, accurate quantification, and is suitable for high-throughput detection.

Description

[0001] This application is a divisional application of patent application 201911372535.0 "6-phosphogluconate dehydrogenase mutants and their use in the preparation of a digitoxin detection reagent" filed on December 27, 2019. TECHNICAL FIELD

[0002] The present application relates to the field of biological detection, in particular to a mutant enzyme 6-phosphogluconate dehydrogenase (abbreviated as G6PDH) and its application in a digitoxin detection kit. BACKGROUND

[0003] Hapten, certain small molecules (molecular weight less than 4000 Da), which alone cannot induce immune response, that is, not immunogenic, but when it is cross-linked or combined with macromolecular proteins or non-antigenic polylysine carrier, it can obtain immunogenicity and induce immune response. These small molecules can bind to response effect products and have antigenicity, which only has immunoreactivity and not immunogenicity, also known as incomplete antigen.

[0004] Hapten can bind to corresponding antibodies to produce antigen-antibody reaction, and cannot alone stimulate the production of antibodies in human or animal body. It only has immunoreactivity and not immunogenicity, also known as incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs belong to haptens. If the haptens are combined with a certain protein molecule (carrier) by chemical methods, new immunogenicity can be obtained, and the corresponding antibodies can be stimulated in animals. Once the haptens are combined with the protein, it forms an antigen cluster of the protein. Some chemical active groups with specific structures (such as penicillin and sulfonamides) with smaller molecular weight than general haptens are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites that can be used for sandwich method, so they cannot be determined by double antibody sandwich method, and are mostly used in competition mode. The principle is that the antigens in the sample and a certain amount of enzyme-labeled antigens compete for binding to the solid-phase antibody. The more the amount of antigens in the sample, the less the enzyme-labeled antigens bound to the solid phase, and the lighter the color. Small molecule hormones, drugs, etc. ELISA determination mostly uses this method.

[0006] The structure of digitoxin (Digitoxin, DG) is as follows:

[0007]

[0008] Digitoxin belongs to a class of naturally occurring cardiac glycosides (CG), which is obtained from purple foxglove, wool foxglove or other suitable digitalis.

[0009] Digitalis glycosides are absorbed rapidly and completely in the gastrointestinal tract, metabolized slowly in the body, and metabolized in the liver, most of the metabolites are inactive. Digoxin is often used for heart failure, indications for congestive heart failure and arrhythmia, due to its slow and long-lasting effect, especially suitable for long-term use in patients with chronic heart failure. Digitalis glycosides and Na + / K + -ATPase reversibly bind to the cell membrane, preventing the enzyme from binding to ATP, inhibiting the active transport of Na + and K + , increasing the intracellular Na + and reducing K + , which is the direct electrophysiological effect and toxicity of digitalis glycosides.

[0010] The amount of digitalis glycosides should be carefully adjusted according to the individual needs of the patient. The therapeutic steady-state plasma concentration of digitalis glycosides is 10 to 25 ng / mL, and higher concentrations (30 ng / mL) may be associated with toxicity. Therefore, effective monitoring of the concentration of digitalis glycosides in the patient's body is required in clinical practice.

[0011] The currently known detection methods of digitalis glycosides mainly include: chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, these detection methods have many defects, such as chemiluminescence, which has good sensitivity, but requires a special device, which is not conducive to popularization due to high investment cost. In the process of clinical detection and diagnosis, the detection methods of homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetry are mainly used.

[0012] The principle of homogeneous enzyme immunoassay is as follows: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-digitalis glycosides) and non-labeled antigen (digitalis glycosides) compete with quantitative antibody (digitalis glycosides antibody) for binding. When the antibody binds more non-labeled antigen, the enzyme-labeled antigen releases more active, and the enzyme catalyzes the substrate NAD+ to generate more NADH, which can be detected by detecting the absorbance change of NADH at 340 nm wavelength, so as to calculate the content of digitalis glycosides in the liquid.

[0013] The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry methods are often limited in application due to complex preparation process and large batch-to-batch difference. CN102768284A describes a preparation method of a small molecule drug-G6PDH enzyme conjugate. However, the method of the prior art relies on the activation of the reaction groups carried by the small molecule drug itself, and then reacts with the enzyme. Such strategy is difficult to ensure the directional reaction between the small molecule drug and the enzyme, resulting in large batch-to-batch difference. SUMMARY

[0014] In view of the needs in the art, the present application provides a novel 6-phosphogluconate dehydrogenase mutant and its use in the preparation of a digitalin test kit.

[0015] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided. Distinct from the 6-phosphogluconate dehydrogenase mutant of the published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphogluconate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of D306C, G426C, D375C.

[0016] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided, which is selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

[0017] According to some embodiments, a polynucleotide encoding the 6-phosphogluconate dehydrogenase mutant of the present application is provided.

[0018] According to some embodiments, an expression vector comprising the polynucleotide of the present application is provided.

[0019] According to some embodiments, a host cell comprising the expression vector of the present application is provided. The host cell can be prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast).

[0020] According to some embodiments, a conjugate is provided, which is the 6-phosphogluconate dehydrogenase mutant of the present application coupled with a hapten at a molar ratio of 1 :n.

[0021] In some embodiments, 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 embodiments, the 6-phosphogluconate dehydrogenase mutant of the present application is coupled with a hapten at a molar ratio of preferably 1 : 1.

[0023] In some particular embodiments, the molecular weight of the hapten is comprised between 100 Da and 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, 1000, 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 the present application, the skilled person will understand that "hapten" also encompasses forms of derivatives thereof. In order to facilitate the coupling with the 6-phosphogluconate dehydrogenase, for those haptens that do not bear a coupling group (e.g. a group reactive with a thiol group) by themselves (e.g. digitoxin), the hapten can be engineered to bear a linker in order to covalently bind to a thiol group. Thus, in the present application, a hapten derivative refers to a hapten engineered to bear a thiol reactive group.

[0025] The hapten is selected from the group consisting of: small molecule drugs (e.g. antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids.

[0026] Haptens such as, but not limited to, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1,25 dihydroxy vitamin D, folic acid, cardiac glycosides (including digitoxin), enzyme phenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the urinary nicotinic metabolite cotinine, urinary morphine, urinary monohydroxyphenylethylene glycol derivatives, neuropeptide tyrosine, plasma glycoproteins, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocortical hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, reverse triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17a hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1a, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive intestinal peptide, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0027] In particular embodiments, the hapten is digitoxin or a derivative thereof.

[0028] In particular embodiments, the hapten is a digitoxin derivative that bears a thiol-reactive group, such as a maleimide, a bromoacetyl group, a vinyl sulfone, or an aziridine.

[0029] In particular embodiments, the hapten is a digitoxin derivative, as shown in Formula I:

[0030]

[0031] wherein,

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

[0033] In some embodiments, X is a maleimide, a bromoacetyl group, a vinyl sulfone, or an aziridine.

[0034] The skilled person will understand that the X function is to react with the thiol group of 6-phosphogluconate. Covalent binding of maleimide, bromoacetyl, vinylsulfone, aziridine and thiol is to be expected. Although specific particular groups are used in the examples, it is not intended to be limited thereto.

[0035] In some particular embodiments, the digitoxin derivative has a structure selected from the group consisting of:

[0036]

[0037]

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

[0039] In some particular embodiments, the digitoxin derivative has a structure selected from the group consisting of:

[0040]

[0041] According to some embodiments, there is provided a reagent comprising the conjugate of the application.

[0042] According to some embodiments, there is provided the use of the 6-phosphogluconate dehydrogenase mutant of the application for the preparation of a digitoxin detection reagent.

[0043] According to some embodiments, there is provided the use of the conjugate of the application for the preparation of a digitoxin detection reagent.

[0044] In particular embodiments, the detection reagent is selected from the group consisting of: an enzyme-linked immunoassay detection reagent, a chemiluminescent immunoassay detection reagent, a homogeneous enzyme immunoassay detection reagent, a latex-enhanced immunoturbidimetric detection reagent.

[0045] In particular embodiments, the detection reagent is preferably a reagent based on competition detection.

[0046] According to some embodiments, there is provided a digitoxin detection kit comprising:

[0047] - a first reagent comprising a substrate, a buffer and a digitoxin antibody; the substrate being a substrate for 6-phosphogluconate dehydrogenase;

[0048] - a second reagent comprising the conjugate of the application and a buffer;

[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 comprising 10 mM to 500 mM buffer, 10 ng / ml to 50 ng / ml digitoxin.

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

[0052] a first reagent comprising:

[0053] 10 mM to 500 mM buffer,

[0054] 5 mM to 50 mM substrate,

[0055] 10 ng / ml to 10 pg / ml of a 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 preservative;

[0059] a second reagent comprising:

[0060] 10 mM to 500 mM buffer,

[0061] 0.01 pg / ml to 10 pg / ml conjugate according to the present application,

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

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

[0064] 0.1 g / L to 5 g / L preservative.

[0065] In some embodiments, the buffer is selected from one or a combination of: tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbitone 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 7 to 8.

[0066] In some embodiments, the stabilizer is selected from one or a combination of: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably, bovine serum albumin.

[0067] In some embodiments, the surfactant is selected from one or a combination of the following: Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.

[0068] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, PC-300, thiomersal; the azide is selected from: sodium azide, lithium azide.

[0069] In some embodiments, the substrate comprises: 6-phosphogluconate, beta-nicotinamide adenine dinucleotide.

[0070] In some specific embodiments, the digoxin antibody is derived from: mouse, rat, cat, dog, primate, bovine, equine, ovine, camelid, avian, human.

[0071] In some specific embodiments, the digoxin antibody is selected from: monoclonal, polyclonal, recombinant, chimeric, antigen-binding fragment.

[0072] According to some embodiments, there is provided a method of preparing a conjugate, comprising the steps of:

[0073] 1) providing a digoxin derivative according to the present application, especially in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethylsulfoxide);

[0074] 2) providing a 6-phosphogluconate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, the buffer having a pH of 6.0 to 8.0);

[0075] 3) contacting the 6-phosphogluconate dehydrogenase mutant and the digoxin derivative in a molar ratio of 1 :n at 18°C to 28°C for 1 hour to 4 hours (preferably 2 hours to 3 hours) so that the digoxin derivative and the 6-phosphogluconate dehydrogenase mutant are conjugated, to obtain the seed conjugate;

[0076] 4) optionally purifying the seed conjugate, such as desalting treatment, etc., as needed.

[0077] In some embodiments, 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 embodiments, steps 1) and 2) are interchangeable.

[0079] In some specific embodiments, prior to coupling, the 6-phosphogluconate dehydrogenase comprises one or more free thiols, allowing the oriented reaction with the digitoxin.

[0080] Wild-type 6-phosphogluconate dehydrogenase does not contain free thiols, so in some specific embodiments, the 6-phosphogluconate dehydrogenase is genetically engineered so that the amino acid at a specific site (position 306, 375 or 426) is mutated to cysteine, thus bearing a free thiol. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 . Structure of digitoxin.

[0082] Figure 2 . Structure of digitoxin derivative.

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

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

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

[0086] Figure 3D . G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0087] EXAMPLE

[0088] Example 1. Synthesis of digitoxin derivative

[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 aqueous periodate solution (1.0 g of periodate) thereto, and stir at room temperature (18 to 28°C, preferably 20 to 25°C) for 1 hour.

[0092] Remove the residue by filtration, remove the solvent under reduced pressure, and extract with dichloromethane. Dry the organic phase with anhydrous sodium sulfate, and remove the solvent under reduced pressure to obtain compound 2 (white solid, 0.90 g, 90% yield).

[0093] 2. Synthesis of compound 4

[0094] Dissolve compound 2 (900 mg, 1.18 mmol) in 10 ml of dry methanol, add compound 3 (318 mg, 1.0 mmol) to the reaction system, and stir at room temperature for 5 minutes. Add sodium cyanoborohydride (146 mg, 2.32 mmol), and stir at room temperature for about 12 hours.

[0095] Remove the solvent under reduced pressure, and purify directly by column chromatography to obtain compound 4 (white solid, 400 mg, 46%).

[0096] 3. Synthesis of compound 5

[0097] Dissolve compound 4 in 15 ml of dichloromethane, and stir at room temperature under nitrogen for 30 minutes, then add 10 ml of piperidine, and stir at room temperature for 2 hours. Remove the solution under reduced pressure, and purify by column chromatography to obtain compound 5 (500 mg, 85%).

[0098] 4. Synthesis of a digitoxin derivative

[0099] Dissolve compound 5 (88 mg, 0.11 mmol) and compound 6 (17 mg, 0.11 mmol) in 8 mL of DCM, add triethylamine (33 mg, 0.33 mmol) dropwise thereto, then add HATU (50 mg, 0.13 mmol), and stir at room temperature for 2 h to obtain a digitoxin derivative (white solid, 48 mg, 43%).

[0100] Confirm the structure of the product by a conventional method. This example allows a digitoxin to have a group that can bind to an enzyme.

[0101] Example 2. Coupling of a digitoxin derivative to a G6PDH molecule

[0102] The G6PDH-digoxigenin conjugate according to the present application is conjugated in the following manner: the thiol-reactive group (such as but not limited to a maleimide group) on the digoxigenin derivative molecule covalently binds to the thiol group on the G6PDH molecule.

[0103] 1. Solution preparation:

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

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

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

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

[0108] 2. Conjugation operation:

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

[0110] 3. After the above reaction system was shaken at room temperature for 4 h, desalting was performed using a desalting column with the above desalting solution, and the protein peak was collected, and the resulting product was the G6PDH-digoxigenin conjugate.

[0111] Example 3. Preparation of a kit

[0112] A kit for detecting digoxigenin was prepared, which comprises:

[0113] Reagent R1, comprising:

[0114] 50 mM HEPES, pH 7.0

[0115] 10 mM 6-phosphogluconate

[0116] 10 mM β-nicotinamide adenine dinucleotide

[0117] 50 ng / ml of digoxigenin antibody (commercially available antibody, without special limitation)

[0118] 1 g / L bovine serum albumin

[0119] 1 g / L Tween20

[0120] 1 g / L sodium azide;

[0121] Reagent R2, comprising:

[0122] 200 mM Tris buffer, pH 8.0

[0123] 0.1 μg / ml G6PDH-digoxigenin conjugate

[0124] 1 g / L bovine serum albumin

[0125] 1 g / L Tween 20

[0126] 1 g / L sodium azide

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

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

[0129] The above reagents (optionally including quality control, calibrators) are assembled into a digoxigenin homogeneous enzyme immunoassay kit.

[0130] Detection Example

[0131] Table 1. Full-automatic biochemical instrument parameters

[0132] Model Hitachi 7180 Analysis point [Rate-A]

[10]

[25]

[34] Wavelength (SUB / MAIN)

[410] /

[340] S.VIL

[10] S.R1; S.R3

[150] ；

[50] ABS. LIMIT

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

[0133] Test Example 1. Accuracy, precision, linearity experiment of the kit of the present application (D306C mutant)

[0134] Table 2. Accuracy, precision

[0135]

[0136] Table 3. Linearity

[0137] Measurement 1 Measurement 2 Measurement 3 Mean value 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] Detection Example 2. Recovery experiment

[0139] Using USP digoxigenin pure product added to mixed serum sample, 4 concentrations were prepared, 21 times were measured at each level. The mean and deviation were calculated.

[0140] Table 4. Recovery experiment

[0141]

[0142]

[0143] Test Example 3. Anti-interference of common drugs

[0144] Table 5. Determination results of anti-interference

[0145] Number Interfering substance Concentration (ug / ml) 1 N-acetyl cysteine 150 2 Amitriptyline 2 3 Ampicillin sodium 100 4 K-hydroxybenzenesulfonate (hydroquinone sulfonate potassium salt) 200 5 Oxylamine 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 Paracetamol 200 18 Chlorpromazine 100 19 Methaqualone 50 20 Nortriptyline 1 21 Cefoxitin 2500 22 Cetirizine dihydrochloride 3 23 Cyclosporine 5 24 Desipramine 3 25 Ethylbenzene 50 26 5-(p-hydroxyphenyl-5-phenylhydantoin) 1000 27 Imipramine 6 28 Phenobarbital 500 29 Phenytoin sodium 500 30 Theophylline 100 31 Valproic acid 1000 32 Ethosuximide 1000 33 Primidone 1000 34 Rifampicin 60

[0146] Test Example 4. Cross reactivity

[0147] The digitalis glycoside pure product was dissolved using a buffer solution to prepare digitalis glycoside calibrators with different concentrations. The following concentrations of digitalis glycoside analogs were added to the mixed serum sample, respectively.

[0148] The reagent of the present application was calibrated using the digitalis glycoside calibrators, and the mixed serum sample and the above sample were determined 5 times, respectively. The ratio of the determination value of the above sample to the determination value of the mixed serum sample was the cross reactivity.

[0149] Table 6. Cross reactivity

[0150] Chemical name Reagent of the present application 5.00 ng / ml digitalis glycoside 309.7％ 20 ng / mL isohydric digitalis glycoside 2.43％ 20 ng / mL digoxigenin-Mono 0.08％ 20 ng / mL digoxigenin-bis 1.60％ 20 ng / mL digoxin 1.05％ 750 ug / mL strophanthin 1.85％ 750 ug / mL desacetyl strophanthin 0.33％

[0151] Test Example 5. Batch difference of digitalis glycoside detection kit

[0152] Three batches of reagents (containing D306C mutant) of the present application were calibrated, respectively, and the differences in absorbance changes of different batches were calculated.

[0153] Table 7. Calibration data between batches

[0154]

[0155] Table 8. Comparison between batches

[0156]

[0157] The control kit (which is different from the kit of the present application in that the enzyme is replaced by the A45C mutant in the prior art) was determined according to the same method, and the CV was in the range of 2.9% to 4.5%, which was significantly lower than the kit of the present application.

[0158] Test Example 6. Antibody inhibition rate

[0159] 1. Detection principle of antibody inhibition rate

[0160] When the antibody binds to the G6PDH-digitalis glycoside conjugate, the G6PDH enzyme activity is affected due to steric hindrance, thereby reducing the efficiency of its catalysis of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental groups with and without the addition of the antibody is compared, and this difference represents the inhibition ability of the antibody to G6PDH.

[0161] 2. Reaction system:

[0162] Table 9. Preparation of detection reagent for antibody inhibition rate

[0163]

[0164] 3. Results:

[0165] By comparing the absorbance values of G6PDH-digoxigenin conjugate with and without the addition of antibody, the inhibition of antibody on G6PDH can be obtained.

[0166] Antibody inhibition rate = [1 - (absorbance change value of G6PDH-digoxigenin with antibody / absorbance change value of G6PDH-digoxigenin without antibody)] x 100%.

[0167] Compared with the published mutation site (A45C), the mutant of the present application has a significant improvement in antibody inhibition rate, which can reach more than 32% (G426C: 32%; D375C: 47%), and the highest is 55% (D306C). The inhibition rate of the previously published mutation sites (such as A45C, K55C) is in the range of 30 to 44%.

[0168] Although not limited to a specific theory, it can be partially explained as:

[0169] Compared with the G6PDH mutants (A45C, K55C) in the prior art, the mutation site (i.e., the site where the free thiol group is introduced) in the enzyme mutant of the present application (especially D306C, D375C) is the position where the hapten (such as hormones, small molecule drugs, etc.) is coupled. When the hapten is bound to the hapten-specific antibody at this position, the steric hindrance formed has the greatest impact on the activity of G6PDH enzyme, and at the same time, after introducing the mutation, it also cannot substantially affect the spatial folding of the molecule. Therefore, the position of this mutation site is very important, and it needs to take into account the activity of G6PDH enzyme, the spatial folding of the coupled molecule, and the sufficient exposure of the hapten epitope.

[0170] Since the enzyme mutant has a significant improvement in antibody inhibition rate, after the enzyme mutant is coupled with digoxigenin to prepare a reagent kit, the reagent has a significant performance improvement in batch variation coefficient, linearity, specificity, and other performance aspects.

Claims

1. A method for preparing a coupling agent, comprising the steps of: Provide glucose-6-phosphate dehydrogenase mutant; Provides digitalisin derivatives; The glucose-6-phosphate dehydrogenase mutant is coupled with the digitalisin derivative; 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; The digitalisin derivative is shown in Formula I: in, m is an integer from 1 to 10; X is selected from: maleimide, bromoacetyl, vinyl sulfone, and aziridine.

2. The method for preparing the coupling compound according to claim 1, wherein X is maleimide.

3. The method for preparing the coupling compound according to claim 1, wherein m is an integer from 1 to 6.

4. The method for preparing the coupling compound according to claim 1, comprising the steps of: 1) Provide the aforementioned digitalisin derivative; 2) Provide the glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant and the digitalisin derivative are contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the digitalisin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. Steps 1) and 2) are interchangeable.

5. In the method for preparing the coupling compound according to claim 4, in step 3): The glucose-6-phosphate dehydrogenase mutant and the digitalisin derivative are contacted at 18°C ​​to 28°C for 2 to 3 hours to couple the digitalisin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

6. The method for preparing the coupling compound according to claim 4, wherein: In step 1), the digitoxin derivative is provided in an aprotic solvent; The aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide.

7. The method for preparing the coupling compound according to claim 4, wherein: In step 2), the glucose-6-phosphate dehydrogenase mutant is provided in a buffer solution; The buffer solution is selected from: PBS, Tris, TapS, and TAPSO. The pH of the buffer solution is between 6.0 and 8.

0.

8. The method for preparing the coupling compound according to claim 4 further includes the step of: 4) The conjugate is purified.

9. The method for preparing the coupling compound according to claim 8, wherein: The conjugate was purified by desalting.

10. The method for preparing the coupling compound according to claim 4, wherein: Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306.

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