Preparation method of conjugate

Abstract

This application relates to a method for preparing a conjugate. 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 the Chinese patent application "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Amikacin Detection Reagents" (Application No. 2020100173769) filed on January 8, 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 an amikacin 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 Amikacin is shown below:

[0007]

[0008] Amikacin is an aminoglycoside antibiotic used to treat infections of the urinary tract, lower respiratory tract, abdominal cavity, soft tissue, bone and joint, reproductive system, and other sites caused by Gram-negative bacilli, as well as sepsis. It can cause irreversible ototoxicity, minimal reversible nephrotoxicity, and rare neuromuscular blockade.

[0009] 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.

[0010] Currently known methods for detecting amikacin 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 to operate, has a long cycle, and is expensive. Luminescence immunoassay reagents are expensive, making them unsuitable for routine therapeutic drug testing and even more challenging to promote. 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.

[0011] Existing methods rely on activating the reactive groups of the small molecule drug (amikacin) itself before reacting with the enzyme. This coupling method can result in multiple amikacins being linked to the same glucose-6-phosphate dehydrogenase. Furthermore, it is difficult to ensure consistency in the coupling sites, making it difficult to guarantee a targeted 1:1 reaction between the small molecule drug and the enzyme, leading to significant batch-to-batch variability. Summary of the Invention

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

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

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

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

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

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

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

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

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

[0021] In some specific embodiments, the molecular weight of the hapten is 100Da to 4000Da, for example: 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210 000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

[0022] According to the present application, the skilled artisan will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as amikacin) that do not themselves carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker to facilitate covalent binding with a sulfhydryl group. Therefore, in the present application, a hapten derivative refers to a hapten that has been modified to carry a sulfhydryl reactive group.

[0023] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0024] Haptens include, but are not limited to, 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.

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

[0026] In a specific embodiment, the hapten is an amikacin derivative that carries a sulfhydryl-reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0027] In a specific embodiment, the hapten is an amikacin derivative, as shown in Formula I:

[0028]

[0029] in,

[0030] 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.

[0031] In some specific embodiments, the amikacin derivative has the structure shown in Formula II:

[0032]

[0033] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0034] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing an amikacin detection reagent.

[0035] According to some embodiments, provided is a use of the conjugate of the present application in preparing an amikacin detection reagent.

[0036] 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.

[0037] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0038] According to some embodiments, provided is a use of the conjugate of the present application in preparing an amikacin detection device.

[0039] 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.

[0040] 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.

[0041] According to some embodiments, there is provided an amikacin detection kit comprising:

[0042] - a first reagent, comprising a substrate, a buffer, and an amikacin antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0043] - a second reagent, comprising the conjugate of the present application and a buffer;

[0044] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 μg / ml to 50 μg / ml amikacin (e.g. 0, 3, 5, 10, 20, 30, 35, 40, 45, 50 μg / ml or any value in between); and

[0045] - Optionally, a control comprising 10 mM to 500 mM buffer, 3 μg / ml to 40 μg / ml (e.g. 3, 4, 5, 10, 20, 30, 40 μg / ml or any value in between) amikacin.

[0046] According to one embodiment, an amikacin detection kit is provided, comprising:

[0047] The first reagent comprises:

[0048] 10mM to 500mM buffer,

[0049] 5mM to 50mM substrate,

[0050] 0.01 μg / ml to 10 μg / ml amikacin 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 μg / ml),

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

[0052] 0.1g / L to 5g / L surfactant,

[0053] 0.1g / L to 5g / L preservatives;

[0054] A second reagent comprising:

[0055] 10mM to 500mM buffer,

[0056] 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),

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

[0058] 0.1g / L to 5g / L surfactant,

[0059] 0.1g / L to 5g / L preservatives.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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 and lithium azide.

[0064] In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

[0065] In some specific embodiments, the amikacin antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, or human.

[0066] In some specific embodiments, the amikacin antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment.

[0067] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0068] 1) providing the amikacin derivative according to the present application, in particular providing the amikacin derivative according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0069] 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);

[0070] 3) contacting the 6-phosphate glucose dehydrogenase mutant and the amikacin derivative at a molar ratio of amikacin 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 coupling between the amikacin derivative and the 6-phosphate glucose dehydrogenase mutant to obtain the conjugate;

[0071] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0072] 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the above values; preferably 50.

[0073] In some embodiments, the contact molar ratio of hapten to enzyme in the reaction system is n:1, 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; preferably 50. In some specific embodiments, steps 1) and 2) can be interchanged or carried out in parallel.

[0074] 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 amikacin.

[0075] 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

[0076] Figure 1 .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0077] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0078] Figure 3 .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0079] Figure 4 .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0080] Example

[0081] Example 1. Synthesis of Amikacin Derivatives

[0082]

[0083] According to the above process, amikacin (123 mg, 0.21 mmol) and compound 1 (64 mg, 0.21 mmol) were dissolved in 5 mL of water and stirred at room temperature (18-28° C.) for 5 h. The amikacin derivative (100 mg, 61%) was separated by HPLC.

[0084] The structure of the amikacin derivative was confirmed to be correct by mass spectrometry and nuclear magnetic resonance. In this embodiment, amikacin is provided with a group that can bind to an enzyme.

[0085] Example 2. Coupling of Amikacin Derivatives with G6PDH Molecules

[0086] 1. Coupling method of the present application

[0087] According to the G6PDH-amikacin 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 amikacin derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0088] 1. Dissolve the amikacin derivative prepared in Example 1 in N,N-dimethylformamide (10 mg / ml);

[0089] 2. G6PDH solution: G6PDH mutant was dissolved in 100 mmol PB, 100 mmol NaCl, pH 8.0, 6 mg / ml;

[0090] 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 amikacin derivative;

[0091] 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-amikacin conjugate.

[0092] 2. Control Coupling Method

[0093] Accurately weigh 100 to 300 mg of amikacin and dissolve it in 5 to 15 mL of anhydrous ethanol;

[0094] Add 5 to 15 mL of 10 to 200 mM sodium periodate dropwise to the above solution, shake gently, and stir at room temperature for 0.5 to 2 hours;

[0095] Add 0.5 to 2 M ethylene glycol (0.5 to 1 mL) dropwise and stir at room temperature for 5 to 10 minutes;

[0096] The reaction mixture was added dropwise to 5 to 15 mL of a 2 to 3% G6PDH solution being stirred, and the pH of the solution was adjusted to 9.0 to 9.5. The reaction was continued by stirring for 0.5 to 2 hours, and the pH of the solution was stabilized.

[0097] Add 100 to 200 mg of sodium tetrahydroborate and stir to reduce for 12 to 24 hours;

[0098] The G6PDH-amikacin conjugate was purified by G-25 gel chromatography column.

[0099] Example 3. Preparation of kit

[0100] The following kit for detecting amikacin was prepared, comprising:

[0101] Reagent R1, containing:

[0102] Tris buffer 100 mM, pH 7.0

[0103] 10 mM glucose 6-phosphate

[0104] 10 mM β-nicotinamide adenine dinucleotide

[0105] 0.5 μg / ml amikacin antibody (commercially available antibody, no special restrictions)

[0106] 1g / L bovine serum albumin

[0107] 1g / L Tween80

[0108] 1g / L sodium azide;

[0109] Reagent R2, including:

[0110] MES buffer 200 mM, pH 8.0

[0111] 0.1 μg / ml G6PDH-amikacin conjugate

[0112] 100mM NaCl

[0113] 1g / L bovine serum albumin

[0114] 1g / L Tween80

[0115] 1g / L sodium azide;

[0116] Calibrator: 20 mM HEPES buffer, and 0 μg / ml, 3 μg / ml, 10 μg / ml, 20 μg / ml, 35 μg / ml, 50 μg / ml amikacin (or added as needed);

[0117] Quality control: 20 mM HEPES buffer, and 4-5 μg / ml, 14-16 μg / ml, 28-32 μg / ml amikacin (or add as needed).

[0118] The above reagents (optionally including quality control products and calibrators) are assembled into an amikacin homogeneous enzyme immunoassay kit.

[0119] Test example

[0120] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, the enzyme-labeled antigen (such as G6PDH-amikacin) and the unlabeled antigen (amikacin) compete with a quantitative antibody (amikacin antibody) for binding. The more the antibody binds to the unlabeled antigen, the more activity the enzyme-labeled antigen releases, and the more NADH the enzyme catalyzes the substrate NAD+ to generate.

[0121] By detecting the absorbance change of NADH at a wavelength of 340nm, the content of amikacin in the liquid can be calculated.

[0122] Table 1. Parameters of fully automatic biochemical analyzer

[0123] Detection model Abbott C16000 Analysis / Time / Read Point Speed / 10min / 28-33 R1 / R2 / S 150：50：3 Wavelength (secondary / primary) 405 / 340 Reaction type Increment Calibration type Spine Calibration points 6 Calibrator concentration 0 / 3 / 10 / 20 / 35 / 50

[0124] Test Example 1. Performance of the kit of this application

[0125] 1. Calibration Experiment

[0126] Table 2. Absorbance of the calibration of the Amikacin Detection Kit

[0127]

[0128] 2. Precision Experiment

[0129] Table 3. Total imprecision

[0130]

[0131] 3. Repeatability

[0132] Table 4. Repeatability

[0133]

[0134]

[0135] 4. Recycling test

[0136] Table 5. Recovery data

[0137]

[0138] 5. Linearity Experiment

[0139] Table 6. Linearity

[0140]

[0141]

[0142] Test Example 2. Accelerated Stability

[0143] After the reagent of the present application (G426 mutant) was accelerated at 37°C for 7 days, the absorbance of the calibration decreased by less than 5%, while after the control reagent was accelerated at 37°C for 7 days, the absorbance of the calibration decreased significantly.

[0144] Table 7. Accelerated stability at 37°C

[0145]

[0146] Test Example 3. Antibody Inhibition Rate

[0147] 1. Principle of Antibody Inhibition Rate Detection

[0148] When the antibody binds to the G6PDH-amikacin 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.

[0149] 2. Reaction system

[0150] Table 8. Preparation of antibody inhibition rate detection reagents

[0151]

[0152] 3. Results

[0153] By comparing the absorbance values ​​of the G6PDH-amikacin conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0154] Antibody inhibition rate = (1 - absorbance change of G6PDH-amikacin conjugate in the presence of antibody / absorbance change of G6PDH-amikacin in the absence of antibody) × 100%.

[0155] Compared to the previously published mutation site (A45C), the mutants described in this application showed significant improvements in enzyme activity retention, reaching over 39% (G426C: 39%; D375C: 48%), and reaching a maximum of 60% (D306C). When the previously published mutation sites (e.g., A45C and K55C) were prepared into G6PDH-amikacin conjugates according to the methods described in this application, the inhibition rates were only 32% and 38%.

[0156] 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.

[0157] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the reagent kit, after the enzyme mutant is coupled with amikacin, has a significant improvement in performance in terms of inter-batch coefficient of variation, linearity, repeatability, stability, etc.

Claims

1. A method for preparing a conjugate, comprising the steps of: 1) Provide amikacin derivatives; 2) Provide 6-phosphate glucose dehydrogenase mutants; 3) the 6-phosphate glucose dehydrogenase mutant is coupled with the amikacin derivative; The amikacin derivative is shown in Formula I: Formula I, in, m is an integer from 1 to 10; Compared to wild-type 6-phosphate glucose dehydrogenase, the 6-phosphate glucose dehydrogenase mutant comprises a D306C or D375C mutation; The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No. 2 or SEQ ID No.

3.

2. The method according to claim 1, wherein: m is an integer of 1 to 5.

3. The method according to claim 1, comprising the steps of: 1) Provide amikacin derivatives; 2) Provide 6-phosphate glucose dehydrogenase mutants; 3) contacting the 6-phosphate glucose dehydrogenase mutant and the amikacin derivative at 18° C. to 28° C. for 1 to 4 hours, so that the amikacin derivative and the 6-phosphate glucose dehydrogenase mutant are coupled to obtain the conjugate; Step 1) and step 2) can be interchangeable or performed in parallel; Before step 3), the 6-phosphate glucose dehydrogenase mutant has a free sulfhydryl group at position 306 or 375.

4. The method according to claim 3, comprising the steps of: 1) providing the amikacin derivative in an aprotic solvent; 2) providing the 6-phosphate glucose dehydrogenase mutant in a buffer; 3) contacting the 6-phosphate glucose dehydrogenase mutant and the amikacin derivative at 18° C. to 28° C. for 2 to 3 hours to allow the amikacin derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate; 4) purifying the conjugate; The buffer is selected from one or a combination of the following: PBS, Tris, TAPS, TAPSO, and the pH of the buffer is 6.0 to 8.0; The aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide. The method according to claim 4 , wherein in step 4), the conjugate is desalted.

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