Gentamycin detection kit

Abstract

This application relates to a gentamicin detection kit. Specifically, the 6-glucose phosphate dehydrogenase mutant described herein comprises one or a combination of the following mutations compared to wild-type 6-glucose phosphate dehydrogenase: D306C, D375C, and G426C. The gentamicin detection kit prepared using the 6-glucose phosphate dehydrogenase mutant described herein has strong specificity, high sensitivity, ease of use, short detection time, and minimal inter-batch variability, and has promising application prospects.

Description

[0001] This application is a divisional application of the patent application with application number 2020100097712 filed on January 6, 2020 and entitled "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Gentamycin Detection Reagent". Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a mutant enzyme 6-phosphate glucose dehydrogenase (abbreviated as G6PDH) and its application in a gentamicin 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] Gentamicin is a mixture of three compounds, the structures of which are shown below:

[0007]

[0008] Gentamicin, first discovered by Weinstein in 1963, is a multi-component aminoglycoside antibiotic produced by Micromonospora bacteria. It includes components C1, C2, C1a, C2a, and C2b. Gentamicin's main components, C1, C2, and C1a, are widely used clinically. C1a, the precursor for the synthesis of azithromycin, has the highest antibacterial activity, followed by C2b, also known as sapacrylomycin. This class of antibiotics binds to the 16S rRNA on the 30S subunit of the bacterial ribosome, causing misreading of the genetic code and thereby blocking bacterial protein synthesis. Therefore, it is primarily used to treat bacterial infections, particularly those caused by Gram-negative bacteria.

[0009] Gentamicin is an aminoglycoside that acts on bacterial ribosomes, inhibiting bacterial protein synthesis and disrupting the integrity of bacterial cell membranes. Gentamicin is rapidly and completely absorbed after intramuscular injection. A certain amount of the drug can also be absorbed through the body surface after local irrigation or topical application. Very little is absorbed into ocular tissues or the systemic blood circulation after ocular administration. Absorption is minimal after oral administration. Following intramuscular injection or intravenous infusion, peak plasma concentrations are reached 30-60 minutes later, with an average peak plasma concentration of approximately 4 μg / ml. Gentamicin has a very low protein binding rate, and after absorption, the drug is primarily distributed in the extracellular fluid. The half-life in adults is 2-3 hours; it may be shortened in patients with fever, anemia, severe burns, or those taking carbenicillin concomitantly. Gentamicin is not metabolized in the body and is primarily excreted in the urine via glomerular filtration.

[0010] Currently known methods for detecting gentamicin include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLI), high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and gas chromatography-mass spectrometry (GC-MS). However, these methods all have significant drawbacks. For example, while chemiluminescence offers excellent sensitivity, it requires specialized equipment, resulting in high costs and hindering widespread adoption. In clinical diagnostics, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetry are the primary methods used.

[0011] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, the enzyme-labeled antigen (such as G6PDH-gentamicin) and the unlabeled antigen (gentamicin) compete with the quantitative antibody (gentamicin 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 from the substrate NAD+ to generate NADH. By detecting the absorbance change of NADH at a wavelength of 340nm, the content of gentamicin in the liquid can be inferred.

[0012] The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to their complex preparation processes and large batch differences.

[0013] The prior art (for example, but not limited to CN108107203A) describes a gentamicin derivative-G6PDH conjugate and its preparation method:

[0014] 1) Weigh G6PDH and dissolve it in PBS buffer at room temperature;

[0015] 2) Dissolve a certain amount of gentamicin, 1-ethyl-3-carbodiimide, and N-hydroxysulfosuccinimide in Mes solution and stir at room temperature for 15-60 minutes for activation;

[0016] 3) Add the activated gentamicin solution dropwise to the dissolved G6PDH and stir to dissolve;

[0017] 4) Stir and dissolve at 2-8°C overnight;

[0018] 5) Purify the coupled enzyme-labeled antigen to obtain glucose dehydrogenase-gentamicin conjugate and store it at 2-8°C.

[0019] However, existing methods rely on activating the reactive groups on the small molecule drug itself before reacting with the enzyme. This strategy makes it difficult to ensure a targeted 1:1 reaction between the small molecule drug and the enzyme, resulting in large batch-to-batch variability. Summary of the Invention

[0020] In view of the needs in the field, the present application provides a novel 6-phosphate glucose dehydrogenase mutant and its use in preparing a gentamicin detection kit.

[0021] 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, D375C, and G426C.

[0022] 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.

[0023] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0024] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0025] 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).

[0026] 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:x. In some embodiments, x is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some specific embodiments, the 6-phosphate glucose dehydrogenase mutant of the present application and the hapten in a molar ratio of preferably 1:1.

[0027] 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.

[0028] 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 gentamicin) 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.

[0029] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0030] Haptens include, but are not limited to, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digitoxin), zymophenolic 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 Disodium 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.

[0031] In specific embodiments, the hapten is gentamicin or a derivative thereof.

[0032] In a specific embodiment, the hapten is a gentamicin derivative bearing a sulfhydryl reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0033] In a specific embodiment, the hapten is a gentamicin derivative, as shown in Formula I:

[0034]

[0035] 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.

[0036] In some embodiments, X is maleimide, bromoacetyl, vinyl sulfone, or aziridine.

[0037] Those skilled in the art will appreciate that the function of X is to react with the sulfhydryl group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine and sulfhydryl groups is contemplated. Although specific specific groups are employed in the examples, this is not intended to be limiting.

[0038] In some specific embodiments, the gentamicin derivative has a structure selected from the following formula:

[0039]

[0040]

[0041] m is an integer of 0 to 20, preferably an integer of 1 to 10, and more preferably an integer of 1 to 6.

[0042] In some specific embodiments, the gentamicin derivative has a structure selected from the following formula:

[0043]

[0044]

[0045] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0046] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a gentamicin detection reagent.

[0047] According to some embodiments, there is provided use of the conjugate of the present application in preparing a gentamicin detection reagent.

[0048] 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.

[0049] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0050] According to some embodiments, there is provided a use of the conjugate of the present application in preparing a gentamicin detection device.

[0051] 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.

[0052] 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.

[0053] According to some embodiments, a gentamicin detection kit is provided, comprising:

[0054] - a first reagent comprising a substrate, a buffer and a gentamicin antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0055] - a second reagent, comprising the conjugate of the present application and a buffer;

[0056] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 μg / ml to 10 μg / ml gentamicin; and

[0057] - Optionally, a quality control comprising 10 mM to 500 mM buffer and 0.5 μg / ml to 8 μg / ml gentamicin.

[0058] According to one embodiment, a gentamicin detection kit is provided, comprising:

[0059] The first reagent comprises:

[0060] 10mM to 500mM buffer,

[0061] 5mM to 50mM substrate,

[0062] 0.1μg / ml to 10μg / ml gentamicin antibody,

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

[0064] 0.1g / L to 5g / L surfactant,

[0065] 0.1g / L to 5g / L preservatives;

[0066] A second reagent comprising:

[0067] 10mM to 500mM buffer,

[0068] 0.1 μg / ml to 10 μg / ml of the conjugate according to the present application,

[0069] 0.1g / L to 5g / L stabilizer,

[0070] 0.1g / L to 5g / L surfactant,

[0071] 0.1g / L to 5g / L preservatives.

[0072] In some embodiments, the buffer is selected from one or a combination of the following: 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 100 mM; the pH of the buffer is 7 to 8.

[0073] 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.

[0074] In some embodiments, the surfactant is selected from one or a combination of the following: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0075] 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.

[0076] In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

[0077] In some specific embodiments, the gentamicin antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, or human.

[0078] In some specific embodiments, the gentamicin antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment.

[0079] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0080] 1) providing a gentamicin derivative according to the present application, in particular providing a gentamicin derivative according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0081] 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);

[0082] 3) contacting the 6-phosphate glucose dehydrogenase mutant and the gentamicin derivative (for example, at a molar ratio of 1:n) at 18° C. to 28° C. for 1 to 4 hours (preferably 2 to 3 hours) to allow the gentamicin derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0083] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0084] 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120; preferably 20 to 40.

[0085] In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0086] 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 gentamicin.

[0087] 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

[0088] Figure 1 .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0089] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0090] Figure 3 .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0091] Figure 4 .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0092] Example

[0093] Example 1. Synthesis of gentamicin derivatives

[0094]

[0095] Where m is 1.

[0096] Gentamycin (100 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, preferably 20-25°C) for 5 h. Direct HPLC separation yielded a gentamicin derivative (110 mg, 78%). The product structure was confirmed by conventional methods. This example provides gentamicin with a group capable of binding to an enzyme.

[0097] Example 2. Coupling of gentamicin derivatives with G6PDH molecules

[0098] 1. Testing Methods of This Application

[0099] According to the G6PDH-gentamicin 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 gentamicin derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0100] 1. Dissolve the gentamicin derivative prepared in Example 1 in DMF (10 mg / ml);

[0101] 2. Provide glucose-6-phosphate dehydrogenase solution (5 mg / mL enzyme, 100 mmol PB, 100 mmol NaCl, pH = 8.0);

[0102] 3. Add 2 ml of glucose 6-phosphate dehydrogenase solution, 7.5 ml of PB solution and 0.5 ml of gentamicin derivative solution and shake at room temperature (18 to 28°C, preferably 20 to 25°C) for 4 h;

[0103] 4. Treatment with a desalting column (desalting solution: 100 mM PB, 0.1% NaN3, 1% NaCl, pH = 8.0) to collect the protein peak and obtain the G6PDH-gentamicin conjugate.

[0104] 2. Control Coupling Method

[0105] Glucose-6-phosphate dehydrogenase-gentamicin conjugate was prepared according to the method of CN108107203A and stored at 2 to 8°C.

[0106] Example 3. Preparation of kit

[0107] Prepare the following kit for detecting gentamicin, comprising:

[0108] Reagent R1, containing:

[0109] 50 mM HEPES, pH 7.0

[0110] 10 mM glucose 6-phosphate

[0111] 10 mM β-nicotinamide adenine dinucleotide

[0112] 1 μg / ml gentamicin antibody (commercially available antibody, no special restrictions)

[0113] 1g / L bovine serum albumin

[0114] 1g / L Tween20

[0115] 1g / L sodium azide;

[0116] Reagent R2, including:

[0117] 200 mM Tris buffer, pH 8.0

[0118] 1 μg / ml G6PDH-gentamicin conjugate

[0119] 1g / L bovine serum albumin

[0120] 1g / L Tween 20

[0121] 1g / L sodium azide;

[0122] Calibrator: 20 mM HEPES buffer, and 0.0, 0.5, 1.5, 3.0, 6.0, 10.0 μg / ml gentamicin (or add as needed);

[0123] Quality control: 20 mM HEPES buffer, and 2.0 μg / ml, 4.0 μg / ml, 7.5 μg / ml gentamicin (or add as needed).

[0124] The above reagents (optionally including quality control products and calibrators) are assembled into a gentamicin homogeneous enzyme immunoassay kit.

[0125] Test example

[0126] Table 1. Parameters of fully automatic biochemical analyzer

[0127]

[0128] Test Example 1. Accuracy, precision, and linearity test of the test kit of this application

[0129] Table 2. Accuracy and precision (for D306C mutant)

[0130]

[0131]

[0132] Table 3. Linearity (for D306C mutant)

[0133]

[0134] Test Example 2: Anti-interference of Common Drugs

[0135] The following compounds were selected as interfering substances and no statistically significant interference was observed in the determination of gentamicin calibrators in the presence of the interfering substances at the concentrations shown in Table 4.

[0136] Table 4. Anti-interference test results (for D306C mutant)

[0137]

[0138]

[0139] Detection Example 3. Correlation

[0140] 1. Test methods

[0141] 80 fresh serum samples were collected and divided into two aliquots, each with a volume of no less than 500 μl. One sample was measured twice using the reagent of this application (specific for the D306C mutant) on a Hitachi 7180 instrument, and the other sample was measured using a Shimadzu HPLC. Correlation analysis was performed using scatter plots of the values ​​measured by the two methods.

[0142] 2. Test results:

[0143] The resulting function is y = 1.0009x - 0.0132, and the correlation coefficient R 2 =0.9963.

[0144] The results showed that the concentration of gentamicin in the sample determined by the reagent of the present application had a good correlation with the concentration of gentamicin in the sample determined by HPLC (which can be regarded as the gold standard).

[0145] Table 5. Correlation analysis (unit: ng / ml)

[0146]

[0147]

[0148] Test Example 4. Inter-batch variation of the gentamicin test kit

[0149] Three batches of the reagent of the present application (D306C mutant) and the reagent prepared by the control coupling method were used for calibration, and the differences in absorbance changes between different batches were calculated.

[0150] Table 6. Calibration data between batches

[0151]

[0152]

[0153] Table 7. Comparison between batches

[0154]

[0155] Test Example 5. Antibody Inhibition Rate

[0156] 1. Principle of Antibody Inhibition Rate Detection

[0157] When the antibody binds to the G6PDH-gentamicin 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.

[0158] 2. Reaction system

[0159] Table 8. Preparation of antibody inhibition rate detection reagents

[0160]

[0161]

[0162] 3. Results

[0163] By comparing the absorbance values ​​of the G6PDH-gentamicin conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0164] Antibody inhibition rate = (1 - absorbance change of G6PDH-gentamicin in the presence of antibody / absorbance change of G6PDH-gentamicin in the absence of antibody) × 100%.

[0165] Compared to the previously published mutation site (A45C), the mutants of this application have significantly improved antibody inhibition rates, reaching over 35% (G426C: 35%; D375C: 48%), and up to 50% (D306C). Previously published mutation sites (such as A45C and K55C) have inhibition rates of 32% and 37%.

[0166] 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.

[0167] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the enzyme mutant and gentamicin conjugate are formulated into a kit, and the reagent has significant performance improvements in terms of inter-batch coefficient of variation, linearity, specificity, etc.

[0168] Test Example 6. Alternatives

[0169] Referring to the preparation method of Example 3, different test kits and control kits were prepared respectively, with the only difference being that the kit prepared in Example 3 was replaced by the following:

[0170] Solution 1: The buffer in the first and second reagents is replaced with phosphate buffer, glycine buffer, borate buffer, or MOPS buffer in the range of 50 to 100 mM pH 7.0-8.0;

[0171] Solution 2: The stabilizer in the first and second reagents was replaced with 0.5 to 2.5 g / L trehalose, sucrose, mannitol, or polyethylene glycol 6000;

[0172] Scheme 3: The surfactant in the first and second reagents was replaced with 0.5 to 2.5 g / L Triton X-100, Tween 80, Brij 35, or Brij 23;

[0173] Scheme 4: The preservative in the first and second reagents is replaced with lithium azide or PC-300;

[0174] Scheme 5: Substitution of the compound of formula II with compounds of formula III, IV and V.

[0175] Three different batches of test kits and control kits in each of the above schemes were tested according to the method of Detection Example 4. The comparison results were close to those in Tables 6 and 7, indicating that the inter-batch variation of the test kits was smaller than that of the control kits (data not shown).

Claims

1. A gentamicin detection kit comprising: - A first reagent comprising: an anti-gentamicin antibody, a substrate, and a buffer: - a second reagent comprising: a buffer and a conjugate; in, The anti-gentamicin antibody is derived from any of the following: mouse, rat, cat, dog, primate, cattle, horse, sheep, camelid, bird, or human; The anti-gentamicin antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment; The conjugate is formed by coupling a 6-phosphate glucose dehydrogenase mutant with a gentamicin derivative in a molar ratio of 1:1; The gentamicin derivative has a structure shown in Formula I: in m is an integer from 1 to 10; X is selected from the group consisting of maleimide, bromoacetyl, vinyl sulfone, and aziridine; Compared to the wild-type 6-phosphate glucose dehydrogenase, the 6-phosphate glucose dehydrogenase mutant comprises any one mutation selected from the group consisting of: D306C, D375C, and G426C; The 6-phosphate glucose dehydrogenase mutant is selected from any one of the following sequences: SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No.

4.

2. The gentamicin detection kit according to claim 1, further comprising a quality control product and / or a calibrator; wherein: The quality control product contains 0.5 μg / ml to 8 μg / ml gentamicin; The calibrators contained 0 μg / ml to 10 μg / ml gentamicin.

3. The gentamicin detection kit according to claim 1, wherein m is an integer from 1 to 6.

4. The gentamicin detection kit according to claim 1, wherein: - A first reagent comprising: 10mM to 500mM buffer, pH 5.0 to 8.5, 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 0.1μg / ml to 10μg / ml anti-gentamicin monoclonal antibody, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, 0.1g / L to 5g / L preservatives; - a second reagent comprising: 10mM to 500mM buffer, pH 5.0 to 8.5, 0.1 μg / ml to 10 μg / ml of the conjugate, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, 0.1g / L to 5g / L preservatives; The buffer is selected from the group consisting of: phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer, and HEPES buffer; The stabilizer is selected from the group consisting of bovine serum albumin, trehalose, sucrose, mannitol, glycerol, glycine, polyethylene glycol 6000, and combinations thereof; The surfactant is selected from the group consisting of Triton X-100, Triton X-405, Tween 80, Tween 20, Brij 35, Brij 23, and combinations thereof; The preservative is selected from the group consisting of: azide compounds, MIT, and biological preservative PC.

5. The gentamicin detection kit according to claim 4, wherein: - A first reagent comprising: 50mM to 200mM buffer, pH 7.0 to 8.0, 10mM to 15mM glucose-6-phosphate, 10mM to 15mM oxidized β-nicotinamide adenine dinucleotide, 0.1μg / ml to 10μg / ml anti-gentamicin monoclonal antibody, 0.5g / L to 2g / L stabilizer, 0.5g / L to 2g / L surfactant, 0.5g / L to 2g / L preservatives; - a second reagent comprising: 50mM to 200mM buffer, pH 7.0 to 8.0, 0.1 μg / ml to 10 μg / ml of the conjugate, 0.5g / L to 2g / L stabilizer, 0.5g / L to 2g / L surfactant, 0.5g / L to 2g / L preservatives.

6. The gentamicin detection kit according to claim 4 or 5, wherein: The azide compound is sodium azide or lithium azide.

7. The gentamicin detection kit according to claim 4 or 5, wherein: The biopreservative PC is PC-300.

8. The gentamicin detection kit according to claim 4 or 5, further comprising a quality control substance and / or a calibrator; wherein: The quality control product contains 10mM to 500mM buffer and 0.5μg / ml to 8μg / ml gentamicin; The calibrators contained 10 mM to 500 mM buffer and 0 μg / ml to 10 μg / ml gentamicin.

9. The gentamicin detection kit according to claim 1 or 4, wherein: The gentamicin derivative is selected from any one of the following structures:

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