Vancomycin Detection Kit

Abstract

This application relates to a vancomycin detection kit. Specifically, the 6-glucose phosphate dehydrogenase mutant of the present application comprises one or a combination of the following mutations compared to wild-type 6-glucose phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the 6-glucose phosphate dehydrogenase mutant of the present application has strong specificity, high sensitivity, convenient operation, short detection time, and accurate quantitative determination, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of 2020100003217 “6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Vancomycin Detection Reagent” filed on January 2, 2020. Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a mutant enzyme glucose-6-phosphate dehydrogenase (G6PDH for short) and its application in a vancomycin detection kit. Background Art

[0003] Haptens are small molecules (molecular weight less than 4000 Da) that cannot induce an immune response on their own, meaning they are not immunogenic. However, when cross-linked or bound to a macromolecular protein or a non-antigenic carrier such as polylysine, they can become immunogenic and induce an immune response. These small molecules can bind to effector products and become antigenic, but are only immunoreactive and not immunogenic, and are also called incomplete antigens.

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate the production of antibodies in humans or animals on its own. It is only immunoreactive and not immunogenic, and is also called an incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically combined with a certain protein molecule (carrier), it will acquire new immunogenicity and can stimulate animals to produce corresponding antibodies. Once a hapten binds to a protein, it forms an antigenic cluster of that protein. Some substances with smaller molecular weight than ordinary haptens but with specific chemically active groups (such as penicillin and sulfonamides) are called simple haptens.

[0005] Small molecule antigens or haptens lack two or more sites for sandwich assays, so the double antibody sandwich assay cannot be used. Instead, a competitive assay is often used. The principle is that the antigen in the specimen competes with a certain amount of enzyme-labeled antigen for binding to the solid-phase antibody. The greater the amount of antigen in the specimen, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This assay is often used for ELISA assays of small molecule hormones, drugs, etc.

[0006] The structural formula of vancomycin is shown below:

[0007]

[0008] Vancomycin has a molecular weight of 1485.71 and is a narrow-spectrum antibiotic that is only effective against Gram-positive bacteria (such as hemolytic streptococci, pneumococci, and enterococci). It is particularly sensitive to drug-resistant Staphylococcus aureus.

[0009] Vancomycin works by inhibiting bacterial cell wall synthesis. It primarily binds to the bacterial cell wall, preventing certain amino acids from entering the cell wall's glycopeptides. It is primarily used clinically to treat severe infections caused by penicillin-resistant Staphylococcus aureus, such as pneumonia, endocarditis, and sepsis. It also has a good effect on infections and sepsis caused by hemolytic streptococci. Vancomycin can also be used to treat colitis and intestinal inflammation. It is also frequently used to prevent infection during the installation of cardiac catheters and intravenous catheters.

[0010] Vancomycin can be used alone or in combination with other antibiotics. There is no cross-resistance between vancomycin and other antibiotics, and drug-resistant strains are rare. It is primarily used for endocarditis, sepsis, and pseudomembranous colitis.

[0011] Vancomycin is not absorbed orally. A single 400mg intravenous infusion achieves a peak plasma concentration of 25.18mg / L immediately after infusion, with an average plasma concentration of 1.90mg / L over 8 hours. Effective plasma concentrations are maintained for 6 to 8 hours. A single 800mg intravenous infusion achieves an average peak plasma concentration of 50.07mg / L. Following intravenous infusion, it is primarily excreted through the kidneys. The average total excretion rate in urine over 24 hours for a single 400mg intravenous infusion is 81.1%, while the average total excretion rate in urine over 24 hours for a single 800mg intravenous infusion is 85.9%.

[0012] Currently known methods for detecting vancomycin include high-performance liquid chromatography (HPLC), luminescent immunoassay, and enzyme-linked immunosorbent assay (ELISA). HPLC requires complex sample pretreatment, is complex and time-consuming, and is expensive. Luminescent immunoassay reagents are expensive, making them unsuitable for routine therapeutic drug testing and hindering widespread adoption.

[0013] The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to their complex preparation processes and large batch differences.

[0014] Prior art CN108717117A describes a vancomycin detection kit. However, this prior art method relies on activating the reactive groups of the small molecule drug (vancomycin) itself before reacting with the enzyme. This coupling method can result in multiple vancomycins being linked to the same glucose-6-phosphate dehydrogenase. Furthermore, it is difficult to ensure consistency in the coupling sites, making it difficult to ensure a targeted 1:1 reaction between the small molecule drug and the enzyme, resulting in significant batch variability. Summary of the Invention

[0015] In view of the needs in the art, the present application provides a novel 6-phosphate glucose dehydrogenase mutant and its use in preparing a vancomycin detection kit.

[0016] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided. Different from the 6-phosphate glucose dehydrogenase mutant disclosed in the previously published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphate glucose dehydrogenase mutant of the present application comprises mutations selected from the group consisting of: D306C, G426C, and D375C.

[0017] 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.

[0018] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0019] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0020] 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).

[0021] According to some embodiments, a conjugate is provided, which is formed by coupling the 6-phosphate glucose dehydrogenase mutant of the present application with a hapten in a molar ratio of 1:n.

[0022] 43, 44, 45, 46, 47, 48, 49, 50.

[0023] In some specific embodiments, the molar ratio of the 6-phosphate glucose dehydrogenase mutant of the present application to the hapten is preferably 1:1.

[0024] 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.

[0025] According to the present application, skilled artisans will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as vancomycin) 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.

[0026] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0027] Haptens include, but are not limited to, vancomycin, 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 thyroid steroids, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin II, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, and osteocalcin.

[0028] In specific embodiments, the hapten is vancomycin or a derivative thereof.

[0029] In specific embodiments, the hapten is a vancomycin derivative that carries a sulfhydryl-reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0030] In a specific embodiment, the hapten is a vancomycin derivative, as shown in Formula I:

[0031]

[0032] In some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

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

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

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

[0036] In a specific embodiment, the detection reagent is selected from: enzyme-linked immunosorbent assay detection reagent, chemiluminescence immunoassay detection reagent, homogeneous enzyme immunoassay detection reagent, latex-enhanced immunoturbidimetric detection reagent.

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

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

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

[0040] In a specific embodiment, the detection device can be prepared in the form of particles (such as latex, magnetic beads), for example, the particles are coated with the reagent according to the present application.

[0041] According to some embodiments, a vancomycin detection kit is provided, comprising:

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

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

[0044] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 μg / ml to 100 μg / ml vancomycin; and

[0045] - Optionally, a quality control comprising 10 mM to 500 mM buffer and 4 μg / ml to 45 μg / ml vancomycin.

[0046] According to one embodiment, a vancomycin detection kit is provided, comprising:

[0047] The first reagent comprises:

[0048] 10mM to 500mM buffer,

[0049] 5mM to 50mM substrate,

[0050] 0.5 to 5 (preferably 1 to 3) mg / L vancomycin antibody,

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

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

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

[0054] A second reagent comprising:

[0055] 10mM to 500mM buffer,

[0056] 10 to 500 (preferably 50 to 200) ng / mL of the conjugate according to the present application,

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

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

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

[0060] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, tris(hydroxymethyl)methane buffer; preferably, phosphate buffer; the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 50 to 100 mM; the pH of the buffer is 7 to 8.

[0061] In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably bovine serum albumin.

[0062] In some embodiments, the surfactant is selected from one or a combination of the following: Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0063] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, biopreservative PC (such as PC-300), thimerosal; the azide is selected from sodium azide and lithium azide.

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

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

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

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

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

[0069] 2) providing a 6-phosphate glucose dehydrogenase mutant, preferably providing the 6-phosphate glucose dehydrogenase mutant in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, wherein the buffer has a pH of 6.0 to 8.0);

[0070] 3) contacting the vancomycin derivative and the 6-phosphate glucose dehydrogenase mutant 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 vancomycin derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

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

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

[0073] In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0074] In some specific embodiments, prior to conjugation, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing for a directed reaction with vancomycin.

[0075] Wild-type 6-phosphate glucose dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific embodiments, 6-phosphate glucose dehydrogenase is genetically engineered so that the amino acid at a specific site (306, 375 or 426) is mutated to cysteine, thereby carrying a free sulfhydryl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0076] Figure 1 .Structure diagram of vancomycin.

[0077] Figure 2 .Structure diagram of vancomycin derivatives.

[0078] Figure 3A .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0079] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0080] Figure 3C .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0081] Figure 3D .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0082] Example

[0083] Example 1. Synthesis of Vancomycin Derivatives

[0084]

[0085] 1. Synthesis of Compound 2

[0086] DCM was added to a round-bottom flask, compound 1 (100 mg, 0.47 mmol) was added thereto, and DMF (2 drops) was added thereto. Oxalyl chloride (90 mg, 0.74 mmol) was slowly added thereto under ice bath and airtight conditions. The reaction system was heated to reflux for 2-4 h, and the solvent was removed under reduced pressure. The excess oxalyl chloride was repeatedly removed with DCM (2-3 times) and used directly in the next step without purification.

[0087] 2. Synthesis of Vancomycin Derivatives

[0088] Vancomycin (100 mg) was dissolved in DMF, and then compound 2 synthesized as above was added to the reaction system. The mixture was stirred at room temperature (18-28°C) for about 2 hours and purified by HPLC to obtain a vancomycin derivative (50 mg, 47%):

[0089]

[0090] The product structure was confirmed by conventional methods.

[0091] This embodiment provides vancomycin with a group that can bind to an enzyme.

[0092] Example 2. Conjugation of vancomycin derivatives with G6PDH molecules

[0093] 1. Coupling method of the present application

[0094] According to the G6PDH-vancomycin 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 vancomycin derivative molecule is covalently bound to the sulfhydryl group on the G6PDH molecule.

[0095] 1. Dissolve the vancomycin derivative prepared in Example 1 in N,N-dimethylformamide (10 mg / ml);

[0096] 2. G6PDH solution: G6PDH (mutant of the present application or mutant of the prior art) was dissolved in 100 mmol PB, 100 mmol NaCl, pH = 8.0;

[0097] 3. Add 200 μl of G6PDH solution to 750 μl of buffer solution (0.05 M Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH = 7.2); then add 50 μl of N,N-dimethylformamide solution of vancomycin derivative;

[0098] 4. The mixed solution was shaken thoroughly at room temperature (18-28°C) for 2-3 hours, desalted, and the protein peak was collected. The obtained product was G6PDH-vancomycin conjugate.

[0099] 2. Control coupling method (refer to the method of CN108717117A)

[0100] Dissolve 5-30 mg of vancomycin in 100-500 μL of dimethylformamide and shake gently to dissolve. At the same time, dissolve 10-40 mg of 100-300 KU of G6PDH in PBS buffer and shake until evenly dissolved.

[0101] The vancomycin solution was slowly added to the G6PDH solution under stirring, and 10-50 μL of glutaraldehyde solution was slowly added dropwise. After the addition was complete, the solution was stirred at 4° C. and reacted overnight.

[0102] The G6PDH-vancomycin conjugate was purified by G-25 gel chromatography and stored at 2-8°C.

[0103] Example 3. Preparation of kit

[0104] The following kit for detecting vancomycin was prepared, comprising:

[0105] Reagent R1, containing:

[0106] 50 mM TAPS, pH 8.0

[0107] 15 mM glucose 6-phosphate

[0108] 15 mM β-nicotinamide adenine dinucleotide

[0109] 2.0mg / L Vancomycin Antibody (commercially available)

[0110] 100mM NaCl

[0111] 1g / L bovine serum albumin

[0112] 1g / L Tween20

[0113] 1g / L sodium azide;

[0114] Reagent R2, including:

[0115] 50 mM Tris buffer, pH 8.0

[0116] 100ng / mL G6PDH-vancomycin conjugate

[0117] 100mM NaCl

[0118] 1g / L bovine serum albumin

[0119] 1g / L Tween 20

[0120] 1g / L sodium azide;

[0121] Calibrators: 20 mM HEPES buffer, and 0 μg / ml, 5 μg / ml, 10 μg / ml, 25 μg / ml, 50 μg / ml, 100 μg / ml vancomycin (or added as needed);

[0122] Quality control: 20 mM HEPES buffer, and 4-8 μg / ml, 15-20 μg / ml, 35-45 μg / ml vancomycin (or add as needed).

[0123] The above reagents (optionally including quality control products and calibrators) are assembled into a vancomycin homogeneous enzyme immunoassay kit.

[0124] Test example

[0125] Principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-vancomycin) and unlabeled antigen (vancomycin) compete for binding with a quantitative antibody (vancomycin antibody). The more the antibody binds to the unlabeled antigen, the more activity the enzyme-labeled antigen releases, and the more NADH is generated by the enzyme-catalyzed substrate NAD+. By detecting the absorbance change of NADH at a wavelength of 340nm, the vancomycin content in the liquid can be inferred.

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

[0127] Detection method Rate method Sample size 2.0 μl Reagent R1 150 μl Reagent R2 50 μl Reading Point 31-34 o'clock Detection wavelength (main) 340 Detection wavelength (sub) 405 Curve fitting method Spline

[0128] Test Example 1. Precision and linearity test of the kit of this application

[0129] 1. Calibration Experiment

[0130] Vancomycin calibrators were dissolved in a buffer solution (0.9% NaCl, 0.1% NaN3) to prepare six different concentrations. The working volume of the calibrator solution was 2-10 μl. Then, 100-200 μl of reagent R1 and 50-100 μl of reagent R2 were added. The rate method was used to detect the primary wavelength at 340 nm and the secondary wavelength at 405 nm. The rate of change in absorbance was read and plotted as a calibration curve. The calibration curves used in this application were generated on a Hitachi 7180, but other mainstream models (such as the AU680 and Abbott C16000) have been tested and are also compatible.

[0131] 2. Precision experiment

[0132] Using the calibration curves established above, high, medium, and low quality control products, as well as clinical samples, were tested 20 times each. As shown in Table 2, the sample test results showed a deviation of less than 3% across 20 replicates.

[0133] Table 2. Precision (for D306C mutant)

[0134]

[0135]

[0136] 3. Linearity Experiment

[0137] Select low-value samples and high-value samples and dilute them according to the arithmetic dilution method. Repeat the test for each sample 3 times. The measured linear data are shown in Table 3. The linear formula is:

[0138] γ=8.8026x+2.9902

[0139] R 2 =0.9985

[0140] Table 3. Vancomycin kit (for D306C mutant) linearity data

[0141] sample Measured value 1 Measured value 2 Measured value 3 mean Theoretical value Relative deviation Absolute deviation 0 1.99 1.99 2.23 2.11 1.84 0.27 1 11.80 10.50 12.50 11.50 11.64 -1.2％ 2 21.93 21.93 21.04 21.49 21.44 0.2％ 3 32.00 31.50 31.80 31.65 31.24 1.3％ 4 41.50 40.21 41.30 40.76 41.03 -0.7％ 5 50.10 51.60 50.90 51.25 50.83 0.8％ 6 58.90 59.75 57.86 58.81 60.63 -3.0％ 7 69.88 71.21 70.85 71.03 70.42 0.9％ 8 80.11 80.34 79.88 80.11 80.22 -0.1％ 9 89.00 90.11 91.12 90.62 90.02 0.7％ 10 98.31 96.55 101.00 98.78 99.81 -1.0％

[0142] Test Example 2. Anti-interference effect on common drugs

[0143] 30 compounds or drugs were selected as interferors. When the vancomycin concentration was about 25 μg / ml and the interferor concentration was 500 μg / ml, the following interferors had no significant interference (for the D306C mutant):

[0144] Acetaminophen, phenacetin, cefazolin, furosemide, benfluazide, kanamycin B, oxytetracycline, hydrochlorothiazide, sisomicin, amikacin, prednisolone, cephalexin, fusidic acid, caffeine, lincomycin, penicillin, ibuprofen, spectinomycin, amphotericin B, prednisone, carbamazepine, gentamicin, cefuroxime, phenytoin, ampicillin, kanamycin A, heparin, salicylic acid, clorazepate.

[0145] Test Example 3: Airborne Stability

[0146] High, medium, and low concentrations of quality control samples were selected and tested three times daily or every other day. The data showed that the calibration period of the vancomycin detection kit (for the 375 mutant) in this application was stable for more than two weeks. After the reagent was opened and placed on the instrument, testing showed that the instrument was stable for more than 40 days (Table 6).

[0147] Test Example 4. Batch Variation of Vancomycin Detection Kit

[0148] 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.

[0149] Table 4. Inter-assay differences

[0150]

[0151] Test Example 5. Antibody Inhibition Rate

[0152] 1. Principle of Antibody Inhibition Rate Detection

[0153] When the antibody binds to the G6PDH-vancomycin 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.

[0154] 2. Reaction system

[0155] Table 5. Preparation of antibody inhibition rate detection reagents

[0156]

[0157] 3. Results

[0158] By comparing the absorbance values ​​of the G6PDH-vancomycin conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0159] Antibody inhibition rate = absorbance change of G6PDH-vancomycin in the presence of antibody / absorbance change of G6PDH-vancomycin in the absence of antibody) × 100%.

[0160] 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: 50%), with a maximum of 56% (D306C). Previously published mutation sites (such as A45C and K55C) had inhibition rates of 32% and 41% respectively.

[0161] 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.

[0162] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the enzyme mutant and vancomycin conjugate are formulated into a kit, and the reagent has significant performance improvements in terms of inter-batch coefficient of variation, linearity, specificity, etc.

[0163]

Claims

1. A vancomycin detection kit comprising: a first reagent comprising a substrate, a vancomycin antibody, and a buffer; a second reagent comprising a conjugate and a buffer; The conjugate is formed by coupling a 6-phosphate glucose dehydrogenase mutant with a vancomycin derivative in a molar ratio of 1:1; Compared to wild-type 6-phosphate glucose dehydrogenase, the 6-phosphate glucose dehydrogenase mutant comprises a D306C or D375C mutation; The 6-phosphate glucose dehydrogenase mutant is represented by the sequence of SEQ ID No. 2 or SEQ ID No. 3; The vancomycin derivative is represented by formula I: Formula I, in, ; m is 1.

2. The vancomycin detection kit according to claim 1, further comprising a calibrator and a quality control product; The calibrator contains 10mM to 500mM buffer, 0μg / ml to 100μg / ml vancomycin; The quality control product contains 10 mM to 500 mM buffer and 2 μg / ml to 60 μg / ml vancomycin.

3. The vancomycin detection kit according to claim 1, comprising: The first reagent comprises: 10mM to 500mM buffer, 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 0.5 mg / L to 5.0 mg / L vancomycin antibodies, 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, 10 ng / mL to 500 ng / 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: TAPS buffer, phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer, and HEPES buffer; The pH of the buffer is 7 to 8; The stabilizer is selected from the group consisting of bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; The surfactant is selected from the group consisting of: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, and AEO7; The preservative is selected from the group consisting of: azide, MIT, biological preservative PC, and thimerosal.

4. The vancomycin detection kit according to claim 3, comprising: The first reagent comprises: 50 mM to 300 mM buffer, 10 mM to 20 mM glucose-6-phosphate, 10 mM to 20 mM oxidized β-nicotinamide adenine dinucleotide, 1.0 mg / L to 3.0 mg / L vancomycin antibodies, 1g / L to 5g / L stabilizer, 1g / L to 5g / L surfactant, 1g / L to 5g / L preservatives; A second reagent comprising: 50 mM to 300 mM buffer, 50 ng / mL to 200 ng / mL of the conjugate, 1g / L to 5g / L stabilizer, 1g / L to 5g / L surfactant, 1g / L to 5g / L preservatives. The vancomycin detection kit according to claim 3 , wherein the preservative is selected from the group consisting of sodium azide, lithium azide, and PC-300.

6. The vancomycin detection kit according to claim 3, wherein: The buffer of the first reagent is TAPS buffer; The buffer of the second reagent is Tris buffer.

7. The vancomycin detection kit according to claim 3, comprising: The first reagent comprises: 50 mM TAPS buffer, pH 8.0, 15mM glucose-6-phosphate, 15mM oxidized β-nicotinamide adenine dinucleotide, 2.0 mg / L vancomycin antibody, 1 g / L bovine serum albumin, 1 g / L Tween20, 1 g / L sodium azide; A second reagent comprising: 50 mM Tris buffer, pH 8.0, 100 ng / mL of the conjugate, 1 g / L bovine serum albumin, 1 g / L Tween20, 1 g / L sodium azide.

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