Use of conjugates in the manufacture of a kit

Abstract

This application relates to the use of conjugates in the preparation of reagent kits. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains a mutation selected from one or a combination of the following, compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Gentamicin detection kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and small batch-to-batch variation, demonstrating promising application prospects.

Description

[0001] This application is a divisional application of application number 2020100097712, filed on January 6, 2020, entitled “Glucose-6-phosphate dehydrogenase mutant and its use in the preparation of gentamicin detection reagents”. 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 a gentamicin 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] 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, including components C1, C2, C1a, C2a, and C2b. The main components C1, C2, and C1a are widely used clinically, with C1a exhibiting the highest antibacterial activity as it is a precursor to etimicin. C2b, also known as sagamycin, is the next most active. These antibiotics bind to the 16S rRNA on the 30S subunit of bacterial ribosomes, causing misreading of the genetic code and thus blocking bacterial protein synthesis. Therefore, they are primarily used to treat bacterial infections, especially those caused by Gram-negative bacteria.

[0009] Gentamicin is an aminoglycoside antibiotic that acts on bacterial ribosomes, inhibiting bacterial protein synthesis and disrupting the integrity of the bacterial cell membrane. Intramuscular injection of gentamicin results in rapid and complete absorption. A certain amount can also be absorbed through the body surface after local irrigation or topical application. Very little is absorbed into the ocular tissues or into the systemic circulation after ocular administration. Oral absorption is minimal. After intramuscular injection or intravenous infusion, peak plasma concentration is reached within 30-60 minutes, with an average peak plasma concentration of approximately 4 μg / ml. Gentamicin has a very low protein binding rate, and after absorption, it is mainly distributed in the extracellular fluid. The half-life in adults is 2-3 hours; this half-life may be shortened in patients with fever, anemia, severe burns, or those concurrently using carbenicillin. 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, high-performance liquid chromatography (HPLC), gas-liquid chromatography (GC), gas chromatography (GC), and mass spectrometry (MS). However, these methods all have several drawbacks. For example, while chemiluminescence immunoassay has good sensitivity, it requires specialized equipment, resulting in high costs and hindering its widespread adoption. In clinical diagnostic testing, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetric assay are the primary methods used.

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

[0012] Existing homogeneous enzyme immunoassay and latex agglutination turbidimetric assays are often limited in application due to their complex preparation processes and large batch-to-batch variations.

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

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

[0015] 2) Dissolve a certain amount of gentamicin, 1-ethyl-3-carbodiimide, and N-hydroxythiosuccinimide in Mes solution and activate by stirring at room temperature for 15-60 minutes.

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

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

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

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

[0020] 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 a gentamicin detection kit.

[0021] 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, D375C, and G426C.

[0022] 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.

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

[0024] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0025] 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).

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

[0027] 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.

[0028] 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., gentamicin) 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.

[0029] Haptens are selected from: small molecule drugs (such as antibiotics and 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 digoxin), 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, free thyroxine, and so on. The following substances are present in the blood: triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfates, 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, and osteocalcin.

[0031] In a specific implementation plan, the hapten is gentamicin or its derivative.

[0032] In a specific implementation, the hapten is a gentamicin derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

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

[0034]

[0035] In some implementations, m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

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

[0037] Those skilled in the art will understand that the function of X is to react with the thiol group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine, and thiol groups is expected. Although specific groups are used in the examples, they are not intended to be limited thereto.

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

[0039]

[0040]

[0041] m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 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 that comprises the conjugate of this application.

[0046] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of gentamicin detection reagents is provided.

[0047] According to some implementation schemes, the use of the conjugates of this application in the preparation of gentamicin detection reagents is provided.

[0048] 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.

[0049] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0050] According to some implementation schemes, the use of the conjugate of this application in the preparation of a gentamicin detection device is provided.

[0051] 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.

[0052] 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 reagent according to this application.

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

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

[0055] - A second reagent, comprising the conjugate and buffer solution of this application;

[0056] -Optionally, the calibrator comprises 10 mM to 500 mM buffer, 0 μg / ml to 10 μg / ml gentamicin; and

[0057] -Optionally, a quality control sample 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 solution

[0061] 5mM to 50mM substrate,

[0062] Gentamicin antibody at concentrations ranging from 0.1 μg / ml to 10 μg / ml

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

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

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

[0066] The second reagent comprises:

[0067] 10mM to 500mM buffer solution

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

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

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

[0071] Preservatives ranging from 0.1 g / L to 5 g / L.

[0072] In some embodiments, the buffer solution is selected from one or a combination of the following: 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 solution is from 10 mmol / L to 500 mmol / L, preferably 100 mM; the pH of the buffer solution is from 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, and 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: azides, MIT, biological preservatives PC (such as PC-300), and thimerosal; the azides are selected from: sodium azide and lithium azide.

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

[0077] In some specific implementation schemes, the gentamicin antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0078] In some specific implementation schemes, the gentamicin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0079] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0080] 1) Provide gentamicin derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0081] 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);

[0082] 3) At 18°C ​​to 28°C, the glucose-6-phosphate dehydrogenase mutant and the gentamicin derivative (e.g., at a molar ratio of 1:n) are contacted for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the gentamicin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

[0083] 4) The conjugate may be purified as needed, for example, by desalting.

[0084] In some embodiments, the contact molar ratio of enzyme to hapten in the reaction system is 1:n, where n is from 1 to 120, 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, 60, 70, 80, 90, 100, 110, 120; preferably 20 to 40.

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

[0086] 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 gentamicin.

[0087] 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

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

[0091] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0092] Example

[0093] Example 1. Synthesis of Gentamicin Derivatives

[0094]

[0095] Where m is 1.

[0096] Gentamicin (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. The gentamicin derivative (110 mg, 78%) was obtained by direct HPLC separation. The product structure was confirmed using conventional methods. This example demonstrates that gentamicin contains a group that can bind to enzymes.

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

[0098] I. Test Methods of This Application

[0099] According to the G6PDH-gentamicin 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 gentamicin derivative molecule is covalently bonded to the thiol 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. Mix 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 hours.

[0103] 4. Desalting column treatment (desalting solution 100mM PB, 0.1% NaN3, 1% NaCl, pH=8.0), collecting protein peaks to obtain G6PDH-gentamicin conjugate.

[0104] II. Contrastive Coupling Method

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

[0106] Example 3. Preparation of the reagent kit

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

[0108] Reagent R1 contains:

[0109] 50mM HEPES, pH 7.0

[0110] 10mM glucose-6-phosphate

[0111] 10mM 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] 1 g / L sodium azide;

[0116] Reagent R2 includes:

[0117] 200mM Tris buffer, pH 8.0

[0118] 1 μg / ml G6PDH-gentamicin conjugate

[0119] 1g / L bovine serum albumin

[0120] 1g / L Tween 20

[0121] 1 g / 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: 20mM HEPES buffer, and 2.0μg / ml, 4.0μg / ml, and 7.5μg / ml gentamicin (or add as needed).

[0124] Assemble the above reagents (optionally including quality control and calibrators) into a gentamicin homogeneous enzyme immunoassay kit.

[0125] Detection example

[0126] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0127]

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

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

[0130]

[0131]

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

[0133]

[0134] Example 2. Common Drug Interference Prevention

[0135] The following compounds were selected as interfering agents. When the interfering agents were present at the concentrations shown in Table 4, the determination of gentamicin calibrators did not show statistically significant interference.

[0136] Table 4. Results of interference resistance assays (for the D306C mutant)

[0137]

[0138]

[0139] Detection Example 3. Correlation

[0140] 1. Test Methods

[0141] Eighty fresh serum samples were collected, each sample was divided into two aliquots, each with a volume of not less than 500 μl. One aliquot was measured twice using the reagents described in this application (for the D306C mutant) on a Hitachi 7180 instrument, while the other aliquot was measured using Shimadzu HPLC. The values ​​obtained by the two methods were analyzed for correlation using scatter plots.

[0142] 2. Experimental Results:

[0143] The resulting function is y = 1.0009x - 0.0132, and the correlation coefficient R0 is... 2 =0.9963.

[0144] The results show that the concentration of gentamicin in the sample determined by the reagents of this application has 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] Example 4. Inter-batch variation of gentamicin detection kit

[0149] Three batches of reagents prepared using the proposed reagent (D306C mutant) and a control reagent were calibrated, and the differences in absorbance variation among the different batches were calculated.

[0150] Table 6. Calibration data between batches

[0151]

[0152]

[0153] Table 7. Comparison between batches

[0154]

[0155] Example 5. Antibody inhibition rate

[0156] 1. Detection principle of antibody inhibition rate

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

[0158] 2. Reaction system

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

[0160]

[0161]

[0162] 3. Results

[0163] By comparing the absorbance values ​​of the G6PDH-gentamicin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0164] Antibody inhibition rate = (1 - change in absorbance of G6PDH-gentamicin with antibody / change in absorbance of G6PDH-gentamicin without antibody) × 100%.

[0165] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in antibody inhibition rate, reaching over 35% (G426C: 35%; D375C: 48%), and up to 50% (D306C). Previously published mutation sites (e.g., A45C, K55C) have inhibition rates of 32% and 37%, respectively.

[0166] 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.

[0167] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the reagent kit formulated by conjugating the enzyme mutant with gentamicin shows a marked improvement in performance in terms of inter-batch coefficient of variation, linearity, and specificity.

[0168] Example 6. Alternative Solution

[0169] Referring to the preparation method of Example 3, different test kits and control kits were prepared, the only difference being that the kits prepared in Example 3 were replaced as follows:

[0170] Option 1: Replace the buffer solutions in the first and second reagents with phosphate buffer, glycine buffer, borate buffer, or MOPS buffer in the range of 50 to 100 mM pH 7.0-8.0.

[0171] Option 2: Replace the stabilizers in the first and second reagents with 0.5 to 2.5 g / L of trehalose, sucrose, mannitol, or polyethylene glycol 6000;

[0172] Option 3: Replace the surfactants in the first and second reagents with 0.5 to 2.5 g / L Triton X-100, Tween 80, Brij 35, or Brij 23;

[0173] Option 4: Replace the preservatives in the first and second reagents with lithium azide or PC-300;

[0174] Option 5: Replace compound II with compounds III, IV and V.

[0175] The test kits and control kits from three different batches in the above schemes were tested according to the method in test example 4. The comparison results were similar to those in Tables 6 and 7, showing that the batch-to-batch variation of the test kits was smaller than that of the control kits (data not shown).

Claims

1. The use of conjugates in the preparation of gentamicin detection reagents, including: The conjugate is formed by conjugating a glucose-6-phosphate dehydrogenase mutant with a gentamicin derivative in a molar ratio of 1:

1. The gentamicin derivative has the structure shown in Formula I: in, m is an integer from 1 to 10; X is selected from any of the following: maleimide, bromoacetyl, vinyl sulfone, aziridine; Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains a mutation selected from any one of the following: D306C, D375C, G426C; The glucose-6-phosphate dehydrogenase mutant is selected from any of the following sequences: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No.

4.

2. The use according to claim 1, wherein the detection reagent is used for homogeneous immunoassay.

3. The use as described in claim 1, wherein: m is an integer from 1 to 6.

4. The use according to any one of claims 1 to 3, wherein: The gentamicin derivative has a structure selected from any of the following:

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