CN116124721A - Gentamicin detection kit - Google Patents

Abstract

The application relates to a gentamicin detection kit. Specifically, the mutant glucose 6-phosphate dehydrogenase of the present application comprises one mutation or a combination thereof selected from the group consisting of: d306C, D375C, G426C. The gentamicin detection kit prepared by using the glucose 6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time and small batch-to-batch difference, and has good application prospect.

Description

Gentamicin detection kit

The application is a divisional application of patent application with the application number 2020100097712 of '6-phosphoglucose dehydrogenase mutant' and application of the mutant in preparing gentamicin detection reagent.

Technical Field

The application relates to the field of biological detection, in particular to mutant enzyme glucose 6-phosphate dehydrogenase (G6 PDH for short) and application thereof in a gentamicin detection kit.

Background

Hapten, some small molecule substances (molecular weight less than 4000 Da) alone are not able to induce an immune response, i.e. are not immunogenic, but are immunogenic when crosslinked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine, inducing an immune response. These small molecule substances can bind to the response effect products, are antigenic, are only immunoreactive, are not immunogenic, and are also called incomplete antigens.

Hapten can bind to the corresponding antibody to generate antigen-antibody reaction, and antigen which can not independently excite human or animal body to generate antibody can not be generated. It is only immunoreactive, not immunogenic, also known as incomplete antigen. Most polysaccharides, lipids, hormones and small molecule drugs belong to the hapten group. If hapten is chemically bound to a protein molecule (carrier), new immunogenicity is obtained and the animal is stimulated to produce the corresponding antibody. Hapten, once bound to a protein, constitutes an antigenic cluster of the protein. Some chemically active substances (such as penicillin, sulfonamides, etc.) which have a smaller molecular weight than the general hapten but a specific structure are called simple haptens.

Small molecule antigens or haptens, which lack two or more sites available for sandwich methods, cannot be assayed by the double antibody sandwich method, and are often in competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with the solid phase antibody. The more the antigen content in the specimen, the less the enzyme-labeled antigen is bound on the solid phase, and the lighter the color development. ELISA assay for small molecule hormones, drugs and the like is commonly used.

Gentamicin (Gentamicin) is a mixture of three compounds, the structures of which are shown below:

gentamicin was first discovered by Weinstein in 1963 as a multicomponent aminoglycoside antibiotic produced by Micromonospora, including C1, C2, C1a, C2a, and C2 b. The main components C1, C2 and C1a of gentamicin are widely used clinically, wherein the highest antibacterial activity is C1a, which is a precursor for synthesizing etimicin, and the next component is C2b, also called sabcomemycin. Such antibiotics are able to bind to 16S rRNA on the 30S subunit of the bacterial ribosome, causing misreading of the genetic code, thus blocking the synthesis of bacterial proteins and therefore are mainly used for the treatment of bacterial infections, in particular infections caused by gram-negative bacteria.

Gentamicin is an aminoglycoside that acts on ribosomes within bacteria, inhibits bacterial protein synthesis, and destroys the integrity of bacterial cell membranes. The absorption is rapid and complete after intramuscular injection of gentamicin. After local washing or local external application, a certain amount of medicine can be absorbed through the surface of the body. Administration via the eye has little absorption into the intraocular tissue or into the systemic blood circulation. The absorption is very little after oral administration. After intramuscular injection or intravenous drip, the blood concentration reaches a peak value for 30-60 minutes, and the average blood concentration peak value is about 4 mug/ml. The protein binding rate of gentamicin is very low, and the drug is mainly distributed in extracellular fluid after absorption. The half-life of adult is 2-3 hours, and the half-life of patients suffering from fever, anemia and severe burn or patients suffering from severe burn by using carbenicillin can be shortened. Gentamicin is not metabolized in the body and is discharged with urine mainly through glomerular filtration.

The currently known gentamicin detection method mainly comprises the following steps: enzyme-linked immunosorbent assay, chemiluminescent immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, the detection methods have more defects, such as chemiluminescence, which has better sensitivity, but needs matched special equipment, so that the detection method has higher input and use cost and is not beneficial to popularization. In the clinical detection and diagnosis process, the homogeneous enzyme immune method (EMIT) and the latex enhanced turbidimetric immunoassay are mainly adopted.

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6 PDH-gentamicin) and unlabeled antigen (gentamicin) compete for being combined with quantitative antibody (gentamicin antibody), when the more the antibody is combined with unlabeled antigen, the more the activity of the enzyme-labeled antigen is released, the more the enzyme-catalyzed substrate NAD+ generates NADH, and the absorbance change of NADH is detected at the wavelength of 340nm, so that the content of gentamicin in the liquid can be deduced.

The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to complex preparation process and large batch-to-batch difference.

In the prior art (for example, but not limited to CN108107203 a) a gentamicin derivative-G6 PDH conjugate and a method for preparing the same are described:

1) Weighing G6PDH, and dissolving in a buffer solution containing PBS at room temperature;

2) Dissolving a certain amount of gentamicin, 1-ethyl-3-carbodiimide and N-hydroxy thiosuccinimide in a Mes solution, stirring and dissolving for 15-60min at room temperature for activation;

3) Dropwise adding the activated gentamicin solution into dissolved G6PDH, and stirring for dissolution;

4) Stirring and dissolving at 2-8deg.C overnight;

5) Purifying the coupled enzyme-labeled antigen to obtain glucose dehydrogenase-gentamicin conjugate, and storing at 2-8 ℃.

However, the prior art methods rely on activation of reactive groups carried by the small molecule drug itself prior to reaction with the enzyme. Such strategies have difficulty guaranteeing orientation between small molecule drugs and enzymes 1:1, resulting in large batch-to-batch variation.

Disclosure of Invention

In view of the needs in the art, the present application provides a novel glucose 6-phosphate dehydrogenase mutant and its use in preparing gentamicin detection kits.

According to some embodiments, a glucose 6-phosphate dehydrogenase mutant is provided. Unlike the mutants of glucose 6 phosphate dehydrogenase of the prior published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose 6-phosphate dehydrogenase mutants of the present application comprise a mutation selected from the group consisting of: d306C, D375C, G426C.

According to some embodiments, there is provided a glucose 6-phosphate dehydrogenase mutant, the glucose 6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

According to some embodiments, a polynucleotide encoding a glucose 6-phosphate dehydrogenase mutant of the present application is provided.

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

According to some embodiments, a host cell is provided comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate which is a glucose 6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: and (3) coupling x. In some embodiments, x is 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some specific embodiments, the molar ratio of the glucose 6-phosphate dehydrogenase mutant to hapten of the present application is preferably 1:1.

in some specific embodiments, the hapten has a molecular weight of 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, 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.

In light of the present application, the skilled artisan will appreciate that "hapten" also includes the form of its derivative. In order to facilitate the coupling with glucose-6-phosphate dehydrogenase, haptens (e.g., gentamicin) that do not themselves have a coupling group (e.g., a group that reacts with a thiol group) may be engineered to have a linker for covalent binding to the thiol group. Thus, in the present application, hapten derivatives refer to haptens engineered to bear a thiol-reactive group.

The hapten is selected from the group consisting of: small molecule drugs (e.g., antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

Hapten such as, but not limited to: theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including digitoxin), zymophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, uronidine metabolite cotinine, uromorphine, uromonophenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactation, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, anti-triiodothyronine, free thyroxine, free triiodothyronine, cortisol urine 17-hydroxycortic steroids, urine 17-ketosterols, dehydroepiandrosterone and sulfates, aldosterone, uronolamine mandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 alpha-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C peptide, gastrin, plasma prostaglandin, plasma 6-keto prostaglandin f1α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, natriuretic acid adenosine cyclophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a specific embodiment, the hapten is gentamicin or a derivative thereof.

In a specific embodiment, the hapten is a gentamicin derivative bearing a sulfhydryl reactive group such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

In a specific embodiment, the hapten is a gentamicin derivative, as shown in formula I:

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, for example 1, 2, 3, 4, 5, 6.

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

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

In some specific embodiments, the gentamicin derivative has a structure selected from the group consisting of:

m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6.

In some specific embodiments, the gentamicin derivative has a structure selected from the group consisting of:

according to some embodiments, there is provided an agent comprising a conjugate of the present application.

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

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

In specific embodiments, the detection reagent is selected from the group consisting of: ELISA detection reagent, chemiluminescent detection reagent, homogeneous ELISA detection reagent and latex enhanced turbidimetry detection reagent.

In a specific embodiment, the detection reagent is preferably a reagent for competition-based detection.

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

In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., 96-well plate), such as a plate coated with reagents according to the present application.

In particular embodiments, the detection device may be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with a reagent according to the present application.

According to some embodiments, there is provided a gentamicin detection kit comprising:

-a first reagent comprising a substrate, a buffer and a gentamicin antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the present application and a buffer;

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

-optionally, a quality control comprising 10mM to 500mM buffer, 0.5 μg/ml to 8 μg/ml gentamicin.

According to one embodiment, there is provided a gentamicin detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer,

5mM to 50mM substrate,

0.1 to 10. Mu.g/ml of gentamicin antibody,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer,

0.1. Mu.g/ml to 10. Mu.g/ml of a conjugate according to the present application,

0.1g/L to 5g/L of stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative.

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 buffer; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 100mM; the pH of the buffer is 7 to 8.

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; bovine serum albumin is preferred.

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 fatty acid diethanolamide, AEO7, preferably Tween20.

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

In some embodiments, the substrate comprises: glucose-6-phosphate, beta-nicotinamide adenine dinucleotide.

In some specific embodiments, the gentamicin antibody is derived from: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans.

In some specific embodiments, the gentamicin antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen binding fragments.

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

1) Providing a gentamicin derivative according to the present application, particularly in an aprotic solvent (such as, but not limited to, acetonitrile, dimethylformamide, dimethylsulfoxide);

2) Providing a glucose 6-phosphate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment such as, but not limited to PBS, tris, TAPS, TAPSO, said buffer having a pH of 6.0 to 8.0);

3) At 18 ℃ to 28 ℃, the glucose 6-phosphate dehydrogenase mutant and the gentamicin derivative (for example, in a molar ratio of 1: n) contacting for 1 to 4 hours (preferably 2 to 3 hours) such that the gentamicin derivative and the glucose 6-phosphate dehydrogenase mutant are coupled to obtain the seed conjugate;

4) The seed conjugate is optionally purified, e.g., desalted, etc., as desired.

In some embodiments, the contacting molar ratio of enzyme to hapten in the reaction system is 1: n, where n is 1 to 120, e.g., 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.

In some specific embodiments, steps 1) and 2) may be interchanged or in parallel.

In some specific embodiments, the glucose 6-phosphate dehydrogenase comprises one or more free sulfhydryl groups prior to coupling, thereby allowing for a directed reaction with gentamicin.

Wild-type glucose 6-phosphate dehydrogenase does not contain a free thiol group, and thus in some specific embodiments, the glucose 6-phosphate dehydrogenase is genetically engineered to have an amino acid mutation at a particular site (306, 375, or 426) to a cysteine, thereby carrying a free thiol group.

Drawings

FIG. 1G 6PDH (wild type) amino acid sequence (SEQ ID No. 1); is derived from Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides.

FIG. 2G 6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3G 6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 4G 6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

EXAMPLE 1 Synthesis of gentamicin derivatives

Wherein m is 1.

Gentamicin (100 mg,0.21 mmol) and compound 1 (64 mg,0.21 mmol) were dissolved in 5mL of water, and stirred at room temperature (18-28 ℃ C., preferably 20-25 ℃ C.) for 5 hours. Direct HPLC isolation gave the gentamicin derivative (110 mg, 78%). The structure of the product was confirmed by a conventional method. This example allows gentamicin to have a group that can bind to enzymes.

EXAMPLE 2 coupling of gentamicin derivative to G6PDH molecule

1. Test method of the application

The G6 PDH-gentamicin conjugate according to the present application was coupled as follows: thiol-reactive groups (such as but not limited to maleimide groups) on gentamicin derivative molecules are covalently bound to thiol groups on G6PDH molecules.

1. The gentamicin derivative prepared in example 1 was dissolved in DMF (10 mg/ml);

2. providing a glucose 6 phosphate dehydrogenase solution (5 mg/mL enzyme, 100mmol PB, 100mmol nacl, ph=8.0);

3. 2ml of glucose 6 phosphate dehydrogenase solution, 7.5ml of PB solution and 0.5ml of gentamicin derivative solution are subjected to shaking reaction at room temperature (18 to 28 ℃, preferably 20 to 25 ℃) for 4 hours;

4. desalting column treatment (desalting solution 100mM PB, 0.1% NaN) ₃ 1% nacl, ph=8.0), protein peaks were collected to give G6 PDH-gentamicin conjugate.

2. Control coupling method

Glucose-6-phosphate dehydrogenase-gentamicin conjugate was prepared by the method described in CN108107203a and stored at 2 to 8 ℃.

EXAMPLE 3 preparation of the kit

The following kit for detecting gentamicin was prepared, which comprises:

reagent R1 comprising:

50mM HEPES，pH 7.0

10mM glucose 6-phosphate

10mM beta-nicotinamide adenine dinucleotide

1. Mu.g/ml gentamicin antibody (commercially available antibody, not particularly limited)

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

200mM Tris buffer, pH 8.0

1 μg/ml G6 PDH-gentamicin conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration material: 20mM HEPES buffer, 0.0, 0.5, 1.5, 3.0, 6.0, 10.0 μg/ml gentamicin (or added as needed);

quality control product: 20mM HEPES buffer, 2.0. Mu.g/ml, 4.0. Mu.g/ml, 7.5. Mu.g/ml gentamicin (or added as needed).

The reagent (optionally including quality control substances and calibrator) is assembled into the gentamicin homogeneous enzyme immunoassay kit.

Test case

TABLE 1 full automatic Biochemical instrument parameters

Detection example 1. Accuracy, precision, and Linear experiments of the kit of the present application

TABLE 2 accuracy and precision (for D306C mutant)

TABLE 3 linearity (for D306C mutant)

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Detection example 2 anti-interference of common drugs

The following compounds were chosen as interferents and gentamicin calibrator was assayed in the presence of interferents at the concentrations indicated in table 4, showing no statistically significant interference.

TABLE 4 measurement results of anti-interference (for D306C mutant)

Detection example 3 correlation

1. Test method

80 fresh serum samples were taken, each divided into two equal parts, each at a volume of not less than 500 μl, one of which was measured twice on a Hitachi 7180 instrument using the reagents of the present application (for D306C mutant) and the other sample was measured using Shimadzu HPLC. Both methods measure correlation analysis using scatter plots.

2. Test results:

the resulting function is y=1.0009 x-0.0132, the correlation coefficient R ² ＝0.9963。

The results show that the concentration value of gentamicin in the reagent measurement sample has good correlation with the concentration value of gentamicin in the HPLC (can be regarded as gold standard) measurement sample.

TABLE 5 correlation analysis (Unit: ng/ml)

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Detection example 4 Gentamicin detection kit batch to batch differences

Three batches of the reagent (D306C mutant) and the reagent prepared by the control coupling method are used for calibrating respectively, and the absorbance change difference of different batches is calculated.

TABLE 6 calibration data between batches

TABLE 7 comparison between batches

Detection example 5 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-gentamicin conjugate, the activity of the G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an added antibody and an experimental group without the added antibody is compared by detecting the change of the NADH amount, and the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. Reaction system

TABLE 8 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-gentamycin conjugate when the antibody is added with the antibody is not added with the antibody, and obtaining the inhibition condition of the antibody on the G6 PDH.

Antibody inhibition = (1-absorbance change for G6 PDH-gentamicin with antibody/absorbance change for G6 PDH-gentamicin without antibody) ×100%.

Compared with published mutation sites (A45C), the mutant has obviously improved antibody inhibition rate, can reach more than 35% (G426C: 35%; D375C: 48%), and can reach up to 50% (D306C). Whereas the inhibition rates of previously published mutation sites (e.g. a45C, K55C) were 32% and 37%.

While not being limited to a particular theory, it may be explained in part as: in comparison with the G6PDH mutant (A45C, K C) in the prior art, the mutation site (i.e. the site for introducing free sulfhydryl) in the enzyme mutant is the coupling site with hapten (such as hormone, small molecule drug, etc.). When hapten is combined with hapten specific antibody at this position, the steric hindrance formed has the greatest effect on the activity of G6PDH enzyme, and after mutation is introduced, the steric folding of the molecule cannot be substantially influenced. Therefore, the position of this mutation site is very important, and it is necessary to combine the activity of the G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The mutant of the enzyme has obvious improvement on the inhibition rate of the antibody. After the conjugate of the enzyme mutant and the gentamicin is prepared into a kit, the reagent has obvious performance improvement in the aspects of the batch variation coefficient, linearity, specificity and the like.

Detection example 6 alternative

Referring to the preparation method of example 3, different test kits and control kits were respectively prepared, except that the kit prepared in example 3 was replaced as follows:

scheme 1: the buffer in the first and second reagents is replaced with phosphate buffer, glycine buffer, boric acid buffer, or MOPS buffer at a PH in the range of 50 to 100mm 7.0-8.0;

scheme 2: the stabilizer in the first and second reagents is replaced with 0.5 to 2.5g/L trehalose, sucrose, mannitol, or polyethylene glycol 6000;

scheme 3: the surfactant in the first and second reagents is replaced with 0.5 to 2.5g/LTriton X-100, tween80, brij35, or Brij 23;

scheme 4: the preservative in the first reagent and the second reagent is replaced by lithium azide or PC-300;

scheme 5: the compound of formula II is replaced with formulas III, IV and V.

Test kits and control kits of three different batches in each of the above schemes were tested with reference to the method of test example 4, and the comparison results were close to tables 6 to 7, showing that the variation in the batch-to-batch difference of the test kit was smaller than that of the control kit (data not shown).

Claims (3)

1. A gentamicin detection kit comprising:

-a first reagent comprising: anti-gentamicin antibody, substrate, buffer:

-a second reagent comprising: buffer, conjugate;

-optionally, a quality control and/or calibrator;

wherein, the liquid crystal display device comprises a liquid crystal display device,

the anti-gentamicin antibody is derived from any one of the following: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans;

the anti-gentamicin antibody is selected from any one of the following: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments;

the quality control comprises 0.5 μg/ml to 8 μg/ml gentamicin;

the calibrator comprises between 0 μg/ml and 10 μg/ml gentamicin;

the conjugate is prepared from a glucose 6-phosphate dehydrogenase mutant and a gentamicin derivative according to a molar ratio of 1:1, coupling;

the gentamicin derivative has a structure shown in a formula I:

wherein the method comprises the steps of

m is an integer from 1 to 10, preferably an integer from 1 to 6;

x is selected from any one of the following: maleimide, bromoacetyl, vinyl sulfone, aziridine;

the glucose 6-phosphate dehydrogenase mutant comprises a mutation selected from any one of the following: d306C, D375C, G426C;

the glucose 6-phosphate dehydrogenase mutant is selected from any one of the following sequences: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

2. The gentamicin detection kit of claim 1 wherein:

-a first reagent comprising:

10mM to 500mM, preferably 50mM to 200mM, buffer,

5mM to 50mM, preferably 10mM to 15mM, glucose-6-phosphate,

5mM to 50mM, preferably 10mM to 15mM, oxidized beta-nicotinamide adenine dinucleotide,

0.1 to 10. Mu.g/ml of an anti-gentamicin monoclonal antibody,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L, of a stabilizer,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L, of surfactant,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L preservative;

-a second reagent comprising:

10mM to 500mM, preferably 50mM to 200mM, buffer,

0.1 to 10. Mu.g/ml of the conjugate,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L, of a stabilizer,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L, of surfactant,

0.1g/L to 5g/L, preferably 0.5g/L to 2g/L preservative;

-optionally, a quality control and/or calibrator;

the buffer is selected from any one of the following: phosphate buffer, glycine buffer, tris buffer, boric acid buffer, MOPS buffer, HEPES buffer;

the buffer pH is 5.0 to 8.5, preferably 7.0 to 8.0;

the stabilizer is selected from any one of the following: bovine serum albumin, trehalose, sucrose, mannitol, glycerol, glycine, polyethylene glycol 6000 or combinations thereof;

the surfactant is selected from any one of the following: triton X-100, triton X-405, tween80, tween20, brij35, brij23 or combinations thereof;

the preservative is selected from any one of the following: azide, MIT, biological preservative PC;

preferably, the azide compound is sodium azide or lithium azide;

preferably, the biological preservative PC is PC-300;

the quality control comprises 10mM to 500mM buffer and 0.5 μg/ml to 8 μg/ml gentamicin;

the calibrator comprises 10mM to 500mM buffer and 0 μg/ml to 10 μg/ml gentamicin.

3. The gentamicin detection kit according to claim 1 or 2, wherein:

the gentamicin derivative has any one structure selected from the following:

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