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] The present application is a divisional application of patent application No. 202010004879.2, filed on January 3, 2020, for "6-phosphogluconate dehydrogenase mutant and its use in preparing digoxin detection reagent". 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 digoxin detection kit. BACKGROUND

[0003] Hapten, certain small molecule substances (molecular weight less than 4000 Da), which alone cannot induce immune response, i.e. not immunogenic, but when it is cross-linked or combined with a large molecular protein or a non-antigenic polylysine carrier, it can obtain immunogenicity and induce immune response. These small molecule substances can be combined with response effect products and have antigenicity, which only has immune reactivity and not immunogenicity, also known as incomplete antigen.

[0004] Hapten can be combined with corresponding antibodies to produce antigen-antibody reaction, and cannot alone stimulate human or animal body to produce antibodies. It only has immune reactivity 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 method, new immunogenicity can be obtained, and the corresponding antibodies can be stimulated in animals. Once the haptens are combined with the protein, an antigen cluster of the protein is formed. Some chemical active groups with specific structure (such as penicillin and sulfonamide) with smaller molecular weight than general haptens are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites 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 the combination with the solid-phase antibody. The more the amount of antigens in the sample, the less the enzyme-labeled antigens combined on the solid phase, and the lighter the color. Small molecule hormones, drugs and other ELISA determinations mostly use this method.

[0006] The structure of digoxin is as follows:

[0007]

[0008] Digoxin is a cardiac glycoside drug, which has positive inotropic effect on the heart, slows the heart rate, and inhibits cardiac conduction, and is used for the treatment of acute and chronic heart failure such as hypertension, valvular heart disease, congenital heart disease, etc. It is especially suitable for heart failure with rapid ventricular rate in atrial fibrillation.

[0009] Common adverse reactions include: proarrhythmia, nausea, vomiting, lower abdominal pain, abnormal weakness, weakness; less common reactions include: blurred vision, central nervous system reactions (such as mental depression or confusion); rare reactions include: drowsiness, headache and skin rash, urticaria (allergic reactions). In 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, ventricular fibrillation, etc.

[0010] Therefore, attention should be paid to the monitoring of adverse reactions of the drug. Moreover, due to the individual differences in drug metabolism, blood drug concentration monitoring should be combined during clinical use to develop a reasonable dosing regimen and try to avoid adverse reactions.

[0011] Currently known methods for detecting digoxin mainly include: high performance liquid chromatography (HPLC), chemiluminescence immunoassay, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, latex agglutination turbidimetry, etc. The HPLC method requires complex sample pretreatment, and is complicated, long in operation period and expensive in cost; the reagent cost of the chemiluminescence immunoassay is high, which is not suitable for routine therapeutic drug detection, and is even more unfavorable for wide application. The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to complex preparation process and large batch-to-batch difference.

[0012] The prior art CN108593905A describes a digoxin detection kit and a preparation method thereof. However, the method of the prior art relies on the activation of the reaction groups carried on the small molecule drug (digoxin) itself, and then reacts with the enzyme. Such coupling method may have multiple digoxins linked on the same glucose-6-phosphate dehydrogenase, and the coupling site is difficult to ensure consistency, which is difficult to ensure the directional 1:1 reaction between the small molecule drug and the enzyme, and leads to large batch-to-batch difference. SUMMARY

[0013] 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 digoxin detection kit.

[0014] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided. Different from the 6-phosphate glucose dehydrogenase mutant of the published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphate glucose dehydrogenase mutant of the present application comprises a mutation selected from the following: D306C, D375C, G426C.

[0015] According to some embodiments, there is provided a 6-phosphogluconate dehydrogenase mutant, which is represented by a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

[0016] According to some embodiments, there is provided a polynucleotide encoding the 6-phosphogluconate dehydrogenase mutant of the present application.

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

[0018] According to some embodiments, there is provided a host cell comprising the expression vector of the present application. The host cell can be prokaryotic (such as bacteria) or eukaryotic (such as yeast).

[0019] According to some embodiments, there is provided a conjugate, which is the 6-phosphogluconate dehydrogenase mutant of the present application coupled with a hapten at a molar ratio of 1 :n.

[0020] 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.

[0021] 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.

[0022] In some specific embodiments, the hapten has a molecular weight of 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, 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.

[0023] According to the present application, the skilled person will understand that "haptene" also encompasses forms of derivatives thereof. For the purpose of coupling to 6-phosphoglucose dehydrogenase, for those haptens which do not carry a coupling group (e.g. a group reactive with a thiol group) themselves, such as digoxin, can be engineered to carry a linker for covalent binding to a thiol group. Thus, in the present application, a haptene derivative means a haptene which has been engineered to carry a thiol reactive group.

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

[0025] The haptene is, for example, but not limited to, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1,25 dihydroxy vitamin D, folic acid, cardiac glycosides (including digoxin, digitoxin), enzyme phenol 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, the neuropeptide tyrosine, plasma cholinesterase, 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, 17 alpha hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1 alpha, prostacyclin, adrenaline, catecholamines, noradrenaline, cholecystokinin, natriuretic, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive intestinal peptide, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0026] In a particular embodiment, the haptene is digoxin or a derivative thereof.

[0027] In a particular embodiment, the haptene is a digoxin derivative which carries a thiol reactive group, such as a maleimide, a bromoacetyl group, a vinylsulfone or an aziridine.

[0028] In a particular embodiment, the haptene is a digoxin derivative, as shown in formula I:

[0029]

[0030] wherein,

[0031]

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

[0033] In some particular embodiments, the digoxin derivative has the structure according to Formula I-1 :

[0034]

[0035] wherein,

[0036]

[0037] According to some embodiments, there is provided a reagent comprising the conjugate of the application.

[0038] According to some embodiments, there is provided the use of the 6-phosphogluconate dehydrogenase mutant of the application for the preparation of a digoxin detection reagent.

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

[0040] 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.

[0041] In particular embodiments, the detection reagent is preferably a reagent based on competition detection.

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

[0043] In particular embodiments, the detection device can be prepared in the form of a well plate (e.g. a 96-well plate), such as a plate coated with the reagent according to the application.

[0044] In particular embodiments, the detection device can be prepared in the form of a particle (e.g. latex, magnetic beads), such as a particle coated with the reagent according to the application.

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

[0046] - a first reagent comprising a substrate, a buffer and a digoxin antibody; the substrate being a substrate for 6-phosphogluconate dehydrogenase;

[0047] - a second reagent comprising the conjugate of the 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 quality 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, there is provided a digoxin detection kit comprising:

[0051] a first reagent comprising:

[0052] 10 mM to 500 mM buffer,

[0053] 5 mM to 50 mM substrate,

[0054] 0.01 pg / ml to 10 pg / 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, 5 pg / ml),

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

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

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

[0058] a second reagent comprising:

[0059] 10 mM to 500 mM buffer,

[0060] 0.01 pg / ml to 10 pg / ml conjugate according to the 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 pg / ml),

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

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

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

[0064] In some embodiments, the buffer is selected from one or a combination of: TAPS, BIS-TRIS, MES, MOPS, phosphoric acid buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbitone buffer, glycine buffer, borate buffer, tris-hydroxymethyl methane buffer; preferably, the buffer is a 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: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably, the stabilizer is bovine serum albumin.

[0066] In some embodiments, the surfactant is selected from one or a combination of: Brij 23, Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, coconut oil fatty acid diethanolamide, AEO7, preferably, the surfactant is Tween 20.

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

[0068] In some embodiments, the substrate comprises: 6-phosphogluconate, beta-nicotinamide adenine dinucleotide.

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

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

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

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

[0073] 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);

[0074] 3) contacting said 6-phosphogluconate dehydrogenase mutant and said digoxin derivative at 18-28°C for 1-4 hours (1, 1.5, 2, 2.5, 3, 3.5, 4 hours, or any value in between, preferably 2-3 hours) at a molar ratio of digoxin derivative: enzyme = 500: 1 to 1: 500 (preferably 50: 1 to 1: 50) to allow coupling of said digoxin derivative and said 6-phosphogluconate dehydrogenase mutant, to obtain said seed conjugate;

[0075] 4) optionally purifying, e.g. desalting, etc., said seed conjugate, as needed.

[0076] In some embodiments, the contacting molar ratio of hapten and enzyme in the reaction system is 1: n, wherein n is 1-500, 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 any range between any of the aforementioned values.

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

[0078] In some specific embodiments, prior to coupling, the 6-phosphogluconate dehydrogenase contains one or more free sulfhydryl groups, allowing directed reaction with digoxin.

[0079] Wild-type 6-phosphogluconate dehydrogenase does not contain free sulfhydryl groups, so in some specific embodiments, the 6-phosphogluconate dehydrogenase is genetically engineered to have an amino acid mutation to cysteine at a specific site (306, 375, or 426), thereby carrying a free sulfhydryl group. BRIEF DESCRIPTION OF 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 3G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

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

[0084] EMBODIMENT

[0085] Example 1. Synthesis of digoxin derivative

[0086]

[0087] 1. Synthesis of compound 2

[0088] Compound 2 (white solid, 0.99 g, 99%) was obtained by dissolving 1.0 g of digoxin in 95% ethanol (80 ml) and then adding a solution of periodic acid (1.0 g) in water (10 ml) thereto, stirring at room temperature (18-28 °C) for 1 hour. The residue was removed by filtration, the solvent was removed under reduced pressure, and dichloromethane was extracted. The organic phase was dried with 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, and stirred at room temperature for 5 minutes. Sodium cyanoborohydride (146 mg, 2.32 mmol) was added, and stirred at room temperature for about 12 hours. The solvent was removed under reduced pressure, and 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, and then 10 ml of piperidine was added, and stirred at room temperature for 2 hours. The solution was removed under reduced pressure, and purified by column chromatography to obtain compound 5 (460 mg, 78%).

[0093] 4. Synthesis of digoxin derivative

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

[0095] 5. The structure of the digoxin derivative is correct without mistake, identified by mass spectrometry and nuclear magnetic resonance.

[0096] This embodiment makes digoxin carry a group that can bind to an enzyme.

[0097] Example 2. Coupling of digoxin derivative with G6PDH molecule

[0098] I. Coupling method of the present application

[0099] According to the G6PDH-digoxin conjugate of the present application, the thiol-reactive group (such as but not limited to a maleimide group) on the digoxin derivative molecule is covalently bound to the thiol 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: dissolve G6PDH (such as the mutant of the present application) in PB 100 mmol, NaCl 100 mmol, 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 N,N-dimethylformamide solution of digoxin derivative to it;

[0103] 4. Shake the above mixed solution well at room temperature (18-28°C) for 2-3 hours, desalt, collect the protein peak, and the resulting product is the G6PDH-digoxin conjugate.

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

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

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

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

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

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

[0110] Purify the G6PDH-digoxin conjugate by G-25 gel chromatography column.

[0111] Example 3. Preparation of the kit

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

[0113] Reagent R1 comprises:

[0114] TAPS buffer 100 mM, pH 7.0

[0115] 15 mM 6-phosphogluconate

[0116] 15 mM β-nicotinamide adenine dinucleotide

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

[0118] 1 g / L bovine serum albumin

[0119] 1 g / L Brij

[0120] 1 g / L sodium azide;

[0121] Reagent R2 comprises:

[0122] Phosphate buffer 200 mM, pH 8.0

[0123] 0.1 μg / ml G6PDH-digoxin conjugate

[0124] 100 mM NaCl

[0125] 1 g / L bovine serum albumin

[0126] 1 g / L Brij

[0127] 1 g / L sodium azide;

[0128] Calibrator: 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 as needed)

[0130] The above reagents (optionally including quality control, calibration) are assembled into a homogeneous enzyme immunoassay kit for digoxin.

[0131] Detection example

[0132] Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-digoxin) competes with non-labeled antigen (digoxin) for binding with quantitative antibody (digoxin antibody). The more the antibody binds with the non-labeled antigen, the more the enzyme-labeled antigen is released, and the more NADH is generated by the enzyme catalyzing the substrate NAD+.

[0133] The absorbance change of NADH is detected at a wavelength of 340 nm, and the content of digoxin in the liquid can be calculated.

[0134] Table 1. Parameters of automatic biochemical analyzer

[0135]

[0136]

[0137] Detection example 1. Performance of the kit of the present application

[0138] 1. Calibration experiment

[0139] Table 2. Calibration absorbance of digoxin detection kit

[0140]

[0141] 2. Precision experiment

[0142] Table 3. Total imprecision

[0143]

[0144] 3. Reproducibility

[0145] Table 4. Reproducibility

[0146]

[0147]

[0148] 4. Recovery test

[0149] Table 5. Recovery data

[0150]

[0151] 5. Linearity experiment

[0152] Table 6. Linearity

[0153]

[0154]

[0155] Detection Example 2. Onboard stability

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

[0157] Table 7. Stability at 37°C acceleration

[0158]

[0159] Detection Example 3. Antibody inhibition rate

[0160] 1. Detection principle of antibody inhibition rate

[0161] When the antibody binds to the G6PDH-digoxin 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 group with and without the addition of the antibody can be compared, which reflects the inhibition ability of the antibody to G6PDH.

[0162] 2. Reaction system

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

[0164]

[0165] 3. Results

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

[0167] Antibody inhibition rate = (absorbance change value of G6PDH-digoxin conjugate with antibody) / (absorbance change value of G6PDH-digoxin conjugate without antibody) x 100%.

[0168] Compared with the published mutation site (A45C), the mutants of the present application have a significant improvement in enzyme activity retention, which can reach more than 40% (G426C: 40%; D375C: 49%), and the highest is 62% (D306C). The published mutation sites (such as A45C, K55C) are prepared into G6PDH-digoxin conjugates according to the method of the present application, and the inhibition rate is only 33% and 40%.

[0169] While not limited to specific theories, this can be partially explained as follows: Compared to existing G6PDH mutants (A45C, K55C), the mutation site (i.e., the site introducing a free thiol group) in the enzyme mutant of this application is the location where it couples with haptens (such as hormones, small molecule drugs, etc.). 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 the sufficient exposure of the hapten epitope.

[0170] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the reagent kit formulated by conjugating the enzyme mutant with digoxigenin shows significant performance improvements in batch-to-batch coefficient of variation, linearity, repeatability, and stability.

Claims

1. A method for preparing a coupling agent, comprising the steps of: 1) Provides digoxin derivatives; 2) Provide a glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant is coupled with the digoxin 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 digoxin derivative is shown in Formula I-1: in, 2. The method according to claim 1, comprising the steps of: 1) Provide the digoxin derivative; 2) Provide the glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant and the digoxigenin derivative are contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the digoxigenin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. Steps 1) and 2) can be interchanged or performed in parallel; Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306.

3. The method according to claim 2, comprising the steps of: 1) The digoxin derivative is provided in an aprotic solvent; 2) Provide the glucose-6-phosphate dehydrogenase mutant in a buffer solution; 3) The glucose-6-phosphate dehydrogenase mutant and the digoxigenin derivative were contacted at 18°C ​​to 28°C for 2 to 3 hours to couple the digoxigenin derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. 4) Purify the conjugate; Steps 1) and 2) can be interchanged or performed in parallel; The buffer solution is selected from: PBS, Tris, TapS, and TAPSO. The pH of the buffer solution is 6.0 to 8.0; The aprotic solvent is selected from: acetonitrile, dimethylformamide, and dimethyl sulfoxide; Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306.

4. The method according to claim 3, in step 4): The conjugate is purified by desalting.

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