Amikacin detection kit

Abstract

This application relates to an amikacin detection kit. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibits high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of Chinese patent application filed on January 8, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of amikacin detection reagent” (application number 2020100173769). 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 an amikacin 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 for Amikacin is shown below:

[0007]

[0008] Amikacin is an aminoglycoside antibiotic used to treat infections caused by Gram-negative bacilli in the urinary tract, lower respiratory tract, abdominal cavity, soft tissues, bones and joints, reproductive system, and sepsis. This product can cause irreversible ototoxicity, but its nephrotoxicity is minimal and reversible; neuromuscular blockade is rare.

[0009] Therefore, monitoring for adverse reactions to this drug should be taken seriously. Furthermore, due to individual differences in drug metabolism, clinical use should incorporate blood drug concentration monitoring to develop a reasonable dosing regimen and minimize the occurrence of adverse reactions.

[0010] Currently known methods for amikacin detection mainly include: high-performance liquid chromatography (HPLC), chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, and latex agglutination turbidimetry. HPLC requires complex sample pretreatment, is complex and time-consuming, and is expensive; chemiluminescent immunoassay reagents are expensive, unsuitable for routine therapeutic drug detection, and hinder widespread adoption. Existing homogeneous enzyme immunoassay and latex agglutination turbidimetry methods are often limited in application due to complex preparation processes and large batch-to-batch variations.

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

[0012] 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 an amikacin detection kit.

[0013] 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.

[0014] 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.

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

[0016] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0017] 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).

[0018] 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.

[0019] 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.

[0020] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 1:1.

[0021] 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.

[0022] 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., amikacin) can be modified to have a linker for covalently binding with a thiol group. Therefore, in this application, a hapten derivative refers to a hapten modified to possess a thiol-reactive group.

[0023] Haptens are selected from: small molecule drugs (such as antibiotics and 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 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, and free thyroxine. Free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfate, 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, osteocalcin.

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

[0026] In a specific implementation, the hapten is an amikacin derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

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

[0028]

[0029] in,

[0030] In some implementations, 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 implementations, the amikacin derivative has the structure shown in Formula II:

[0032]

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

[0034] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of amikacin detection reagents is provided.

[0035] According to some implementation schemes, the use of the conjugate of this application in the preparation of amikacin detection reagents is provided.

[0036] 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.

[0037] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0038] According to some implementation schemes, the use of the conjugate of this application in the preparation of an amikacin detection device is provided.

[0039] In a specific implementation, the detection device can be prepared in the form of a well plate (e.g., a 96-well plate), for example, the plate is coated with the reagent according to this application.

[0040] In a specific implementation, the detection device can be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with the reagents according to this application.

[0041] According to some implementation schemes, an amikacin detection kit is provided, comprising:

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

[0043] - A second reagent, comprising the conjugate and buffer solution of this application;

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

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

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

[0047] The first reagent comprises:

[0048] 10mM to 500mM buffer solution

[0049] 5mM to 50mM substrate,

[0050] Amikacin antibody from 0.01 μg / ml to 10 μg / ml (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] 0.1 g / L to 5 g / L stabilizer

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

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

[0054] The second reagent comprises:

[0055] 10mM to 500mM buffer solution

[0056] 0.01 μg / ml to 10 μg / ml of the conjugates according to this 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] 0.1 g / L to 5 g / L stabilizer

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

[0059] Preservatives ranging from 0.1 g / L to 5 g / L.

[0060] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, 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 is from 10 mmol / L to 500 mmol / L, preferably from 50 to 100 mM; the pH of the buffer is from 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, and 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: azides, MIT, biological preservatives PC (such as PC-300), and thimerosal; the azides are selected from: sodium azide and lithium azide.

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

[0065] In some specific implementations, the amikacin antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0066] In some specific implementation schemes, the amikacin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0067] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0068] 1) Provide amikacin derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0069] 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);

[0070] 3) At 18°C ​​to 28°C, the glucose-6-phosphate dehydrogenase mutant and the amikacin derivative are contacted at a molar ratio of amikacin derivative:enzyme = 500:1 to 1:500 (preferably 50:1 to 1:50) for 1 hour to 4 hours (1, 1.5, 2, 2.5, 3, 3.5, 4 hours or any value between them, preferably 2 hours to 3 hours) to couple the amikacin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate;

[0071] 4) The conjugate may be purified as needed, for example, by desalting.

[0072] In some embodiments, the contact molar ratio of hapten to enzyme in the reaction system is 1:n, where n is from 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 any range 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, where n is from 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 any range of the above values; preferably 50. In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0074] 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 amikacin.

[0075] 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

[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 The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0079] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0080] Example

[0081] Example 1. Synthesis of Amikacin Derivatives

[0082]

[0083] Following the above procedure, 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. Amikacin derivatives (100 mg, 61%) were obtained by HPLC separation.

[0084] Mass spectrometry and nuclear magnetic resonance analysis confirmed the correct structure of the amikacin derivative. This embodiment demonstrates that amikacin contains a group that can bind to enzymes.

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

[0086] I. Coupling Method of This Application

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

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

[0089] 2. G6PDH solution: The G6PDH mutant was dissolved in 100 mmol PB and 100 mmol NaCl at pH 8.0 at a concentration of 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 to it;

[0091] 4. The above mixed solution is shaken thoroughly at room temperature (18-28℃) for 2-3 hours to desalt it, and the protein peak is collected. The resulting product is G6PDH-amikacin conjugate.

[0092] II. Contrastive 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 to the above solution, shake gently, and stir at room temperature for 0.5 to 2 hours;

[0095] Add 0.5 to 1 mL of 0.5 to 2 M ethylene glycol dropwise, and stir the reaction at room temperature for 5 to 10 minutes;

[0096] Add the above reaction mixture dropwise to 5 to 15 mL of a 2 to 3% G6PDH solution that is being stirred, adjust the pH of the solution to 9.0 to 9.5, continue stirring the reaction for 0.5 to 2 hours, and allow the pH of the solution to stabilize.

[0097] Add 100 to 200 mg of sodium borohydride 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 the reagent kit

[0100] Prepare the following kit for detecting amikacin, comprising:

[0101] Reagent R1 contains:

[0102] Tris buffer 100mM, pH 7.0

[0103] 10mM glucose-6-phosphate

[0104] 10mM 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] 1 g / L sodium azide;

[0109] Reagent R2 includes:

[0110] MES buffer 200mM, 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] 1 g / L sodium azide;

[0116] Calibrators: 20 mM HEPES buffer, and amikacin at concentrations of 0 μg / ml, 3 μg / ml, 10 μg / ml, 20 μg / ml, 35 μg / ml, and 50 μg / ml (or add as needed);

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

[0118] Assemble the above reagents (optionally including quality control and calibrators) into an amikacin homogeneous enzyme immunoassay kit.

[0119] Detection example

[0120] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-amikacin) and unlabeled antigen (amikacin) compete to bind with a quantitative amount of antibody (amikacin antibody). The more the antibody binds to the unlabeled antigen, the more activity of the enzyme-labeled antigen is released, and the more NADH is generated from the substrate NAD+ by the enzyme.

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

[0122] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0123] Testing models Abbott C16000 Analysis / Time / Reading Point Rate / 10min / 28-33 R1 / R2 / S 150：50：3 Wavelength (sub- / primary) 405 / 340 reaction type Increasing Calibration type Spine Calibration point 6 Calibrator concentration 0 / 3 / 10 / 20 / 35 / 50

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

[0125] 1. Calibration Experiment

[0126] Table 2. Calibration absorbance 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. Recycling Data

[0137]

[0138] 5. Linear Experiment

[0139] Table 6. Linear

[0140]

[0141]

[0142] Example 2. Accelerated stability testing

[0143] The absorbance of the reagent (G426 mutant) accelerated at 37℃ for 7 days decreased by less than 5%, while the absorbance of the control reagent decreased significantly after 7 days of acceleration at 37℃.

[0144] Table 7. Accelerated stability at 37℃

[0145]

[0146] Example 3. Antibody inhibition rate

[0147] 1. Detection principle of antibody inhibition rate

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

[0149] 2. Reaction system

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

[0151]

[0152] 3. Results

[0153] By comparing the absorbance values ​​of the G6PDH-amikacin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0154] Antibody inhibition rate = (1 - change in absorbance of G6PDH-amikacin conjugate with antibody / change in absorbance of G6PDH-amikacin without antibody) × 100%.

[0155] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in enzyme activity retention, reaching over 39% (G426C: 39%; D375C: 48%), and up to 60% (D306C). When published mutation sites (e.g., A45C, K55C) were prepared into G6PDH-amikacin conjugates using the method described in this application, the inhibition rates were only 32% and 38%, respectively.

[0156] 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.

[0157] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the formulation of a kit by conjugating the enzyme mutant with amikacin showed significant performance improvements in batch-to-batch coefficient of variation, linearity, repeatability, and stability.

Claims

1. An amikacin detection kit, comprising: The first reagent comprises: substrate, amikacin antibody, and buffer solution; The second reagent comprises: a conjugate and a buffer solution; The conjugate is formed by conjugating a glucose-6-phosphate dehydrogenase mutant with an amikacin derivative in a molar ratio of 1:

1. The amikacin derivative is shown in Formula II: Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains a D306C or D375C mutation; The glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No. 2 or SEQ ID No.

3.

2. The amikacin detection kit according to claim 1, comprising: The first reagent comprises: 10mM to 500mM buffer solution 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 0.01 μg / ml to 10 μg / ml amikacin antibody, 0.1 g / L to 5 g / L stabilizer 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L of preservatives; The second reagent comprises: 10mM to 500mM buffer solution The conjugate was prepared at concentrations from 0.01 μg / ml to 10 μg / ml. 0.1 g / L to 5 g / L stabilizer 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L of preservatives; The buffer solutions in the first and second reagents are each independently selected from: TAPS buffer, phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer, and HEPES buffer; The buffer solutions in the first and second reagents each have an independent pH of 7 to 8; The stabilizers in the first reagent and the second reagent are each independently selected from: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; The surfactants in the first reagent and the second reagent are each independently selected from: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, and AEO7; The preservatives in the first reagent and the second reagent are each independently selected from: azide, MIT, biological preservative PC, and thimerosal.

3. The amikacin detection kit according to claim 2, comprising: The first reagent comprises: 50mM to 300mM buffer solution 10mM to 20mM glucose-6-phosphate, 10mM to 20mM oxidized β-nicotinamide adenine dinucleotide, 0.1 μg / ml to 1 μg / ml amikacin antibody, 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives; The second reagent comprises: 50mM to 300mM buffer solution The conjugate was prepared at concentrations of 0.05 μg / ml to 0.5 μg / ml. 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives.

4. The amikacin detection kit according to claim 2, comprising: The first reagent comprises: 50mM TAPS buffer, pH 8.0 10mM glucose-6-phosphate, 10mM oxidized β-nicotinamide adenine dinucleotide, 0.5 μg / ml amikacin antibody, 1g / L bovine serum albumin, 1g / L Tween20 1 g / L sodium azide; The second reagent comprises: 50mM Tris buffer, pH 8.0 0.1 μg / ml of the conjugate, 1g / L bovine serum albumin, 1g / L Tween20 1 g / L sodium azide.

5. The amikacin detection kit according to claim 2, wherein the preservative is selected from: sodium azide, lithium azide, and PC-300.

6. The amikacin detection kit according to claim 1, further comprising a calibrator containing 10 mM to 500 mM buffer and 0 μg / ml to 50 μg / ml amikacin.

7. The amikacin detection kit according to claim 1, further comprising a quality control, said quality control comprising 10 mM to 500 mM buffer and 3 μg / ml to 40 μg / ml amikacin.

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