Glucose-6-phosphate dehydrogenase mutants and their use in the preparation of detection reagents

Abstract

A glucose-6-phosphate dehydrogenase mutant and its use in preparing a detection reagent are disclosed. Compared to the wild-type glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant contains a combination of the following mutations: 56C, 306C, and 454C. A detection kit prepared by using glucose-6-phosphate dehydrogenase has strong specificity, high sensitivity, convenient operation, short detection time, and accurate quantification, making it suitable for high-throughput detection.

Description

[Technical field]

[0001] The present application relates to the field of biological detection, in particular to the enzyme glucose-6-phosphate dehydrogenase (abbreviated as G6PDH) with multiple mutations and its application in detection kits. [Background technology]

[0002] Haptens are a type of low molecular weight substance (having a molecular weight of less than 4000 Da) that cannot induce an immune response by itself and therefore has no immunogenicity. However, by crosslinking or binding with a carrier such as a high molecular weight protein or non-antigenic polylysine, they can acquire immunogenicity and induce an immune response. These low molecular weight substances can bind to response effectors and have antigenicity. Haptens are also known as incomplete antigens, since they are only immunoreactive and not immunogenic.

[0003] Haptens can bind to corresponding antibodies to produce antigen-antibody reactions, but cannot initiate antibody production in the human or animal body by themselves. Most polysaccharides, lipids, hormones, and low-molecular-weight drugs are haptens. When a hapten is combined with a specific protein molecule (carrier) by chemical methods, it acquires new immunogenicity, and animals begin to produce the corresponding antibodies.

[0004] Low molecular antigens (or haptens) cannot be detected by sandwich-ELISA because they do not have more than three sites that can be used in the sandwich method, and competitive mode is generally used to detect haptens. The principle is that the antigen in the sample competes with a certain amount of enzyme-labeled antigen for binding to the solid-phase antibody. The higher the antigen content in the sample, the less enzyme-labeled antigen will bind to the solid phase, and the lighter the color will be. This method is mainly used in ELISA determination of low molecular hormones and drugs.

[0005] The currently known hapten detection methods mainly include radioimmunoassay, enzyme-linked immunoassay, chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography, and LC-MS. However, all of these detection methods have many disadvantages, for example, the isotopes used in radioimmunoassay have many disadvantages such as radioactive contamination, short stability period, and inconvenience of operation, and enzyme-linked immunoassay is cumbersome and time-consuming, which makes it unsuitable for clinical use. Chemiluminescence has good sensitivity, but requires special equipment and is expensive to use, which makes it unsuitable for general use. The main methods in the clinical detection and diagnosis process are enzyme-amplified immunoassay technique (EMIT) and latex-enhanced turbidimetric immunoassay.

[0006] The principle of homogeneous enzyme immunoassay is that in a homogeneous liquid reaction system, enzyme-labeled antigen competes with unlabeled antigen for binding with a certain amount of antibody, and the more antibody that binds to the unlabeled antigen, the higher the activity released by the enzyme-labeled antigen and the more NADH produced by the enzyme substrate NAD+. By detecting the change in absorbance at a wavelength of 340 nm, the content of hapten in the liquid can be estimated.

[0007] The methods in the prior art rely on activating the reactive group of a hapten (such as a small molecule drug) and reacting it with an enzyme. In such a conjugation method, some small molecule drugs may be conjugated to a single glucose-6-phosphate dehydrogenase, but it is difficult to ensure the consistency of the conjugation site, so it is difficult to ensure a 1:1 directional reaction between the small molecule drug and the enzyme, resulting in high batch-to-batch variability. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 6,090,567 [Patent Document 2] China Publication No. 110174363 Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the need in the art, the present application provides novel glucose-6-phosphate dehydrogenase mutants and their use in the preparation of detection reagents. [Means for solving the problem]

[0010] According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant. The glucose-6-phosphate dehydrogenase mutant of the present application comprises a combination of the following mutations compared to the wild type: 56C, 306C, and 454C.

[0011] The mutants in the present application are different from the glucose-6-phosphate dehydrogenase mutants in published patents, such as U.S. Pat. No. 6,090,567 (Homogeneous Immunoassay Using Mutant Glucose-6-phosphate Dehydrogenase), and also different from the glucose-6-phosphate dehydrogenase mutants disclosed in China Publication No. 110174363 (comprising a single mutation selected from D306C, D375C, or G426C compared to the wild type).

[0012] According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant as set forth in SEQ ID NO:2.

[0013] According to some embodiments, a polynucleotide is provided that encodes a glucose-6-phosphate dehydrogenase variant of the present application.

[0014] According to some embodiments, an expression vector is provided that comprises a polynucleotide of the present application.

[0015] According to some embodiments, a host cell is provided comprising the expression vector of the present application. The host cell may be a prokaryotic cell (e.g., a bacterium) or a eukaryotic cell (e.g., a yeast).

[0016] According to some embodiments, there is provided a conjugate obtained by conjugating a glucose-6-phosphate dehydrogenase mutant of the present application with a hapten in a molar ratio of 1:m.

[0017] In some embodiments, m is 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

[0018] In certain embodiments, the glucose-6-phosphate dehydrogenase mutants of the present application are directionally conjugated to a hapten, preferably in a 1:3 molar ratio.

[0019] In certain embodiments, the hapten is from 100 Da to 4000 Da, e.g., 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 Da , 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 molecular weight.

[0020] From this application, a person skilled in the art will understand that "hapten" also includes its derivative form. To facilitate conjugation with glucose-6-phosphate dehydrogenase, a hapten that does not possess a conjugation group (e.g., a group that reacts with a sulfhydryl group) can be designed to have a linker so that it can be covalently bonded to a sulfhydryl group. Thus, in this application, a hapten derivative refers to a hapten that has been modified to have a sulfhydryl-reactive group.

[0021] The hapten is selected from the group consisting of small molecule drugs (antibiotics, psychotropic drugs, etc.), hormones, metabolites, sugars, lipids, amino acids, and short peptides (having a molecular weight of less than 4000 kDa or an amino acid length of 50 amino acid residues or less).

[0022] Haptens include, but are not limited to, the following: - Anticancer or antitumor drugs: taxanes, paclitaxel and its derivatives, docetaxel, docetaxel, irinotecan, SN38, topotecan, topotecan hydrochloride, topotecan, cisplatin, carboplatin, oxaliplatin, camptothecin and its derivatives, hydroxycamptothecin, vinblastine, vincristine, emetine, emetine hydrochloride, colchicine, doxorubicin, epirubicin, pirarubicin, valrubicin, doxorubicin or doxorubicin hydrochloride, epirubicin, daunorubicin, daunomycin, mitomycin, aclacinomycin, idamycin, bleomycin, peplomycin, mithramycin, rapamycin, bleomycin, streptozotocin, podophyllotoxin, actinomycin D, maytansinoids, amikacin, mitoxantrone, all-trans retinoid vindesine, vinorelbine, gemcitabine, capecitabine, cladribine, pemetrexed disodium, tegafur, letrozole, anastrozole, fulvestrant, goserelin, triptorelin, leuprolide, buserelin, temozolomide, cyclophosphamide, ifosfamide, gefitinib, sunitinib, erlotinib, icotinib, lapatinib, sorafenib, imatinib, dapagliflozin ... Satinib, nilotinib, sirolimus, everolimus, mercaptopurine, methotrexate, 5-fluorouracil, dacarbazine, hydroxyurea, vorinostat, ixabepilone, bortezomib, cytarabine, etoposide, azacitidine, teniposide, propranolol, procaine, tetracaine, lidocaine, bexarotene, carmustine, chlorambucil, methylbenzidine, thiotepa, - Antibiotics, antivirals, and antifungals: macrolides, defensins, polymyxin E mesylate, polymyxin, polymyxin B, capreomycin, bacitracin, gramicidin, amphotericin B, aminoglycosides, gentamicin, paramecin, tobramycin, kanamycin, aminohydroxybutylkanamycin A, neomycin, streptomycin, nystatin, echinocandins, carbenicillin, penicillin, penicillin-susceptible agents, penicillin G, penicillin V, penicillinase-resistant agents, methicillin, oxaliplat ... Sacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, penem, amoxicillin, vancomycin, daptomycin, anthracycline, chloramphenicol, erythromycin cyclocarbonate, flavomycin, oleandomycin, troleandomycin, clarithromycin, erythromycin, dirithromycin, roxithromycin, nitrogen erythromycin, azithromycin, flurithromycin, josamycin, spiramycin, midecamycin, medegomycin, albomycin, miokamycin , rokitamycin, doxycycline, cinolide A, teicoplanin, lamporanin, midoplanin, chrystine, fluorocytosine, miconazole, econazole, fluconazole, itraconazole, ketoconazole, voriconazole, clotrimazole, bifonazole, netilmicin, amikacin, caspofungin, micafungin, terbinafine, fluoroquinolones, lomefloxacin, norfloxacin, ciprofloxacin, enoxacin, ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, mofetil, cyclosporine ... Xyfloxacin, grepafloxacin, gatifloxacin, sparfloxacin, temafloxacin, pefloxacin, amifloxacin, fleroxacin, tosufloxacin, prulifloxacin, irloxacin, pazufloxacin, clinafloxacin, sitafloxacin, idarubicin, tosufloxacin, ramoplanin, nucleoside antiviral agents, ribavirin, antipseudomonas penicillin, ticarcillin, azlocillin, mezlocillin, piperacillin, ampicillin, hetacillin, gravicillin, amoxicillin, cephalosporins,Cefpodoxime, cefprozil, cephobutene, cefazoxime, ceftriaxone, cefotaxime, cephapirin, cephalexin, cephradine, cefoxitin, cefamandole, cefazolin, ceflorizine, cefaclor, cefadroxil, cephalexin, cefuroxime, cefurate, cefotaxime, cefprozil, cefepime, cefixime, cefonicil, cefoperazone, cefotetan, cefmetazole, ceftazidime, loracarbef, moxalactam, ceftibuten, cephalosporin II, ceftriaxone, cyanoacetyl cepha rosporin, monobactam, aztreonam, carbapenem, imipenem, pentanamidine hydroxyether sulfonate, imipenem, meropenem, pentamidine isothiourea, salbutamol sulfate, lidocaine, oxynaline sulfate, beclomethasone, m-hydroxyisoproterenol sulfate, beclomethasone dipropionate, triamcinolone acetamide, budesonide, budesonide acetonide, fluticasone, ipratropium bromide, flunisolide, sodium cromoglycate, cyclosporine, cyclosporine A, ergotamine tartrate, - Cytochalasin B, aminomethylbenzoic acid, sodium paraaminohippurate, aminoglutethimide, aminolevulinic acid, aminosalicylic acid, pamidronate, amsacrine, anagrelide, anastrozole, levamisole, busulfan, cabergoline, leuprorelin, cilastatin sodium, disodium clodronate, amiodarone, ondansetron, deacetylcycloprochlorometherone, megestrol, testosterone, estramustine, exemestane, fluorohydroxymethyltestosterone, diethylstilbestrol, phenoxyethanol, Xofenadine, fludarabine, fludrocortisone, fluticasone, deferoxamine, flutamide, bicalutamide, thalidomide, L-dopa, leucovorin, lisinopril, levothyroxine sodium, nitrogen mustard, medroxyprogesterone, m-hydroxynorephedrine bitartrate, mexiletine, mitotane, nicotine, nilutamide, octreotide, pentostatin, plicamycin, porfimer, prednisone, procarbazine, pleuropiperazine, raltitrexed, streptozotocin, sirolimus, tacrolimus Mus, tamoxifen, teniposide, tetrahydrocannabinol, thioguanine, thiotepa, dolasetron, granisetron, formoterol, melphalan, midazolam, alprazolam, sumatriptan, low molecular weight heparin, amifostine, carmustine, gemcitabine, lomustine, teixobactin, aspirin, salicylic acid, phenylbutazone, indomethacin, naproxen, diclofenac, meloxicam, nabumetone, etodolac, sulindac, acemetacin, diacerein, amdoxovir, cyanuric blue, arylaminochito , aminocaproic acid, aminophendoperidone, aminolevulinic acid, butylene glycol diester mesylate, chloromethyl bisphosphate, chloromethyl bisphosphate disodium, L-dihydroxyphenylalanine, dichloromethyldiethylamine, m-hydroxylamine bitartrate, o-dichlorobenzenedichloroethane, prochlorperazine, ondansetron, raltitrexed tacrolimus, tamoxifen, tanipsted, tetrahydrocannabinol, aroylhydrazone, sumatriptan, miokamycin, rokitamycin,Phenesterine, piposulfan, epistine hydrochloride, insulin, antisense nucleotides, small RNA, - Vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides, mycophenolic acid, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, nicotine metabolites, urinary morphine, urinary monophenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, trans-triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone Androsterone and sulfate, aldosterone, urinary vanillic 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, epinephrine, catecholamines, norepinephrine, cholecystokinin, naloxine, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0023] In a particular embodiment, the hapten is tobramycin or a derivative thereof.

[0024] Although tobramycin is used as a specific example, a person skilled in the art can understand that the technical effect of the present application is not dependent on a specific type of hapten, and other haptens that are immunologically detectable by competitive methods are applicable.

[0025] In certain embodiments, the hapten is a derivative of tobramycin that has a sulfhydryl reactive group, such as maleimide, bromoacetyl, vinylsulfone, or aziridine.

[0026] In certain embodiments, the hapten has formula I:

[0027] [ka]

[0028] m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6; X is selected from the group consisting of maleimide, bromoacetyl, vinylsulfone, and aziridine; More preferably, X is maleimide. It is a derivative of tobramycin, as shown in

[0029] In certain embodiments, the hapten has formula II:

[0030] [ka] It is a derivative of tobramycin, as shown in

[0031] According to some embodiments, a reagent is provided that includes a conjugate of the present application.

[0032] 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 detection reagent.

[0033] According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a detection reagent.

[0034] In certain embodiments, the detection reagent is selected from the group consisting of an enzyme-linked immunoassay detection reagent, a chemiluminescent immunoassay detection reagent, a homogeneous enzyme immunoassay detection reagent, and a latex-enhanced immunoturbidimetric detection reagent.

[0035] In certain embodiments, the detection reagent is preferably a competitive detection reagent.

[0036] According to some embodiments, - a first reagent comprising a substrate and an antibody against a hapten, the substrate being a substrate for glucose-6-phosphate dehydrogenase; - a second reagent comprising a conjugate of the present application; - optionally a calibrator comprising a buffer and a hapten at 10 mM to 500 mM; - Optionally, quality controls containing 10 mM to 500 mM buffer and hapten. A kit for detecting a hapten is provided, comprising:

[0037] According to one embodiment, 10 mM to 500 mM buffer, 5mM to 25mM substrate, 0.01 μg / L to 1 mg / L of antibodies against the hapten, 10 mM to 300 mM NaCl, 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative A first reagent comprising: 10 mM to 500 mM buffer, 0.01 μg / L to 1 mg / L of the conjugate of the present application; 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative a second reagent comprising A kit for detecting a hapten is provided, comprising:

[0038] According to some specific embodiments, - a first reagent comprising a substrate and an antibody to tobramycin, the substrate being a substrate for glucose-6-phosphate dehydrogenase; - a second reagent comprising a conjugate of the present application; - optionally a calibrator comprising a buffer solution of 10 mM to 500 mM and tobramycin having a known concentration; - Optionally, quality controls containing 10 mM to 500 mM buffer and tobramycin with known concentrations. A kit for detecting tobramycin is provided, comprising:

[0039] According to one embodiment, 10 mM to 500 mM buffer, 5mM to 25mM substrate, 0.01 μg / ml to 10 μg / ml of an antibody against tobramycin; 10 mM to 300 mM NaCl, 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative A first reagent comprising: 10 mM to 500 mM buffer, 0.01 μg / ml to 10 μg / ml of the conjugate of the present application; 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative a second reagent comprising A kit for detecting tobramycin is provided, comprising:

[0040] In some embodiments, the buffer is selected from one or a combination of tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, and trihydroxymethylmethane buffer, preferably phosphate buffer, the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 100 mM, and the pH of the buffer is 6.5 to 8.0, preferably 7.2 or 7.0.

[0041] In some embodiments, the stabilizing agent is selected from one or a combination of bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000, preferably bovine serum albumin.

[0042] In some embodiments, the surfactant is selected from one or a combination of Brij 23, Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, coconut fatty acid diethanolamide, and AEO7, preferably Tween 20.

[0043] In some embodiments, the preservative is selected from one or a combination of azide, MIT, PC-300, and thimerosal, and the azide is selected from sodium azide or lithium azide.

[0044] In some embodiments, the substrate comprises glucose-6-phosphate and β-nicotinamide adenine dinucleotide.

[0045] According to some embodiments, 1) providing a hapten or a derivative thereof according to the present application, in particular providing a hapten or a derivative thereof according to the present application in an aprotic solvent (e.g., but not limited to, acetonitrile, dimethylformamide, dimethylsulfoxide); 2) providing a glucose-6-phosphate dehydrogenase mutant of the present application, preferably in a buffer (providing a reaction environment, for example, but not limited to, PBS, Tris, TAPS, TAPSO, etc., the pH of the buffer being 6.0 to 8.0); 3) contacting the glucose-6-phosphate dehydrogenase mutant with a hapten or a derivative thereof in a molar ratio of 1:n at 18° C. to 28° C. for 1 hour to 4 hours, preferably 2 hours to 3 hours, so that the hapten or a derivative thereof is conjugated with the glucose-6-phosphate dehydrogenase mutant to obtain a conjugate; 4) Optionally, purifying the conjugate as needed, for example by desalting. A method for preparing a conjugate is provided, comprising:

[0046] In some embodiments, n is from 1 to 500, 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, 150, 200, 250, 300, 350, 400, 450, 500, or a range between any of the above values.

[0047] In certain embodiments, the glucose-6-phosphate dehydrogenase variant is contacted with the hapten or derivative thereof at a molar ratio of 1:30 to 1:120, including 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, or 1:120.

[0048] In certain embodiments, steps 1) and 2) are interchangeable or parallel.

[0049] In certain embodiments, glucose-6-phosphate dehydrogenase contains a free sulfhydryl group prior to conjugation, allowing for a directed 1:3 reaction with a hapten or a derivative thereof. [Brief description of the drawings]

[0050] [Figure 1] FIG. 1 shows the G6PDH (wild type) amino acid sequence (SEQ ID NO: 1) from the Leuconostoc species Leuconostoc pseudomesenteroides. [Diagram 2] FIG. 2 shows the G6PDH mutant (SEQ ID NO: 2). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0051] Example 1 Synthesis of tobramycin derivatives. Tobramycin (98 mg, 0.21 mmol) and compound 1 (64 mg, 0.21 mmol) were dissolved in 5 mL of water and stirred at room temperature for 5 hours. The tobramycin derivative was obtained by HPLC separation. The synthetic route was as follows:

[0052] [ka]

[0053] The structure of the synthesized product was confirmed by conventional methods. The effect of this embodiment is to provide a low molecular weight antigen (or hapten) with a group capable of binding to an enzyme, and the technical effect of this application does not depend on a specific hapten derivative.

[0054] Example 2 Preparation of mutants The desired DNA was synthesized, for example, by using well-known genetic engineering methods, inserted into an appropriate expression vector (such as an E. coli expression vector), expressed in an expression host, and purified (such as affinity purification) to obtain the enzyme mutant shown in SEQ ID NO:2.

[0055] Example 3 Conjugation of tobramycin derivatives to G6PDH mutants The G6PDH-tobramycin conjugate of the present application was obtained in the following manner: A sulfhydryl reactive group (such as a maleimide group) of the tobramycin derivative molecule was covalently bound to a sulfhydryl group of the G6PDH molecule.

[0056] 4 μl of a solution (5 mg / mL enzyme, 100 mmol PB, 100 mmol NaCl, pH=8.0) containing the G6PDH enzyme mutant of Example 2 (or a control G6PDH enzyme mutant of the prior art), 200 μl of the PB solution, and 800 μl of the tobramycin derivative prepared in Example 1 were reacted with shaking at room temperature (18 to 28° C., preferably 20 to 25° C.) for 2.5 hours.

[0057] After treatment with a desalting column (desalting solution: 100 mM PB, 0.1% NaN3, 1% NaCl, pH=8.0), the protein peak was pooled to obtain the G6PDH-tobramycin conjugate.

[0058] Example 4 Kit Preparation A kit for the detection of tobramycin was prepared, comprising: 1. Preparation of the first reagent: HEPES buffer 100mM, pH7.0 Anti-tobramycin antibody 0.5μg / ml β-nicotinamide adenine dinucleotide, oxidized form, 15 mmol / L Glucose-6-phosphate 15mmol / L Bovine serum albumin 1g / L Tween 20 1g / L Sodium azide 1g / L. 2. Preparation of the second reagent: PB buffer 100mM, pH8.0 G6PDH-tobramycin conjugate 0.1μg / ml Bovine serum albumin 1g / L Tween 20 1g / L Sodium azide 1g / L. 3. Calibrators: Pure tobramycin was diluted (or added as needed) with buffer solution (100 mM HEPES buffer) to concentrations of 0, 0.6, 2.0, 4.0, 6.0, and 10.0 mg / L. 4.Quality Control: Pure tobramycin was diluted (or added as needed) with buffer solution (100 mM HEPES buffer) to concentrations of 1.5 mg / L, 3 mg / L, and 8 mg / L.

[0059] Test Example

[0060] [Table 1]

[0061] Test Example 1. Calibration absorbance of tobramycin detection kit

[0062] [Table 2]

[0063] Test Example 2. Reproducibility of the tobramycin detection kit High, medium, and low quality controls were determined in replicates of 20. The reproducibility of the kit of the present invention was shown to be favorable, with a CV of less than 2.61%.

[0064] [Table 3]

[0065] Test Example 3. Linearity of the tobramycin detection kit The low and high samples screened were arithmetically diluted. Each sample was tested in triplicate. The average measured concentration and theoretical concentration were analyzed to evaluate the recovery rate, which showed that the deviation of the results was less than 10%, and the linear performance reached 10 μg / ml.

[0066] [Table 4]

[0067] Test Example 4. Accuracy The pure tobramycin product of the United States Pharmacopoeia was dissolved in various concentrations to prepare stock solutions, which were then diluted equally (at least 20 times) with serum to prepare tobramycin solutions with different serum concentrations. Measurements were performed using the kit of the present invention, and the deviation from the theoretical value was calculated. The results showed that the deviation of the recovery rate was less than 6%, and the precision was favorable.

[0068] [Table 5]

[0069] Test Example 5. Antibody Inhibition Rate 1. Principle of antibody inhibition rate detection When the antibody binds to the G6PDH-tobramycin conjugate, it affects the G6PDH enzyme activity through steric hindrance, reducing the efficiency of the enzyme 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, and such a difference reflects the inhibitory ability of the antibody against G6PDH.

[0070] 2. Reaction system

[0071] [Table 6]

[0072] [Table 7]

[0073] 3.Results Inhibition of G6PDH by the antibody could be obtained by comparing the absorbance of the G6PDH-tobramycin conjugate with and without the addition of antibody.

[0074]

number

[0075] Here, ΔA refers to the difference in absorbance between two test time points on the reaction curve.

[0076] [Table 8]

[0077] Without being limited to a particular theory, this can be partially explained by the fact that, compared with the G6PDH mutants of the prior art, the mutation sites (i.e., sites where free sulfhydryl groups are introduced) in the enzyme mutants of the present application (K56C / D306C / D454C) are positions for conjugation with haptens (e.g., hormones, small molecule drugs, etc.). When the hapten binds to a hapten-specific antibody at these positions, the steric hindrance formed has the greatest effect on the activity of the G6PDH enzyme, while at the same time cannot substantially affect the steric folding of the molecule after the mutation is introduced. Therefore, the location of these mutation sites is very important, and it is necessary to consider the activity of the G6PDH enzyme, the spatial folding of the conjugated molecule, and also the appropriate exposure of the hapten epitope.

[0078] The enzyme mutant has obvious advantages in the calibration of absorbance due to the significantly increased antibody inhibition rate. After the conjugate formed by the enzyme mutant and the hapten is formulated into a kit, the performance of the reagent is significantly improved in terms of reproducibility, total imprecision, linearity, and specificity.

Claims

1. A glucose-6-phosphate dehydrogenase mutant comprising a combination of the following mutations compared to a Leuconostoc pseudomesenteroides wild-type glucose-6-phosphate dehydrogenase: 56C, 306C, and 454C, A glucose-6-phosphate dehydrogenase mutant as shown in SEQ ID NO:

2.

2. A polynucleotide encoding the glucose-6-phosphate dehydrogenase mutant of claim 1.

3. An expression vector comprising the polynucleotide of claim 2.

4. A host cell comprising the expression vector of claim 3, A host cell without cells capable of developing into an animal or plant.

5. The host cell of claim 4, selected from the group consisting of prokaryotic cells, yeast cells, and CHO cells.

6. A conjugate obtained by conjugating the glucose-6-phosphate dehydrogenase mutant according to claim 1 with a hapten in a molar ratio of 1:m, m is 1 to 3, the hapten is selected from the group consisting of a small molecule drug, an antibiotic, a hormone, a metabolite, a polysaccharide, a lipid, and a short peptide; A conjugate, wherein the hapten has a molecular weight of between 100 Da and 4000 Da.

7. The conjugate described in claim 6, wherein m is 3.

8. A reagent comprising the conjugate according to claim 6 or 7.

9. In preparing the detection device: A conjugate according to claim 6 or 7, comprising the glucose-6-phosphate dehydrogenase mutant of claim 1. The use of any one of the following: The use, wherein the detection device is a detection device for a hapten.

10. The method of claim 1, wherein the detection device is selected from the group consisting of an enzyme-linked immunoassay detection device, a chemiluminescent immunoassay detection device, a homogeneous enzyme immunoassay detection device, and a latex-enhanced immunoturbidimetric detection device; the hapten is selected from the group consisting of a small molecule drug, an antibiotic, a hormone, an amino acid, a metabolite, a polysaccharide, a lipid, and a short peptide; The hapten has a molecular weight of 100 Da to 4000 Da.

10. The use according to claim 9.

11. The use according to claim 10, wherein the hapten has a molecular weight of from 200 Da to 1500 Da.

12. The use according to claim 9, wherein the detection device is selected from the group consisting of a reagent, a kit, a well plate, a test paper, a magnetic bead, a latex particle, and a chip.

13. a first reagent comprising a substrate, an anti-hapten antibody, and a buffer; A second reagent comprising the conjugate according to claim 6 or 7 and a buffer solution; A detection kit comprising:

14. The detection kit of claim 13, further comprising a calibrator comprising a 10 mM to 500 mM buffer and a hapten having a known concentration.

15. 15. The detection kit of claim 13 or 14, further comprising a quality control comprising a buffer of 10 mM to 500 mM and a hapten with a known concentration.

16. 10 mM to 500 mM buffer, 5mM to 25mM substrate, 0.01μg / L to 1mg / L of anti-hapten antibodies, 10 mM to 300 mM NaCl, 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative A first reagent comprising: 10 mM to 500 mM buffer, 8. The conjugate according to claim 6 or 7, at 0.01 μg / L to 1 mg / L. 0.1g / L to 5g / L of stabilizer, 0.1 g / L to 5 g / L of a surfactant, and 0.1g / L to 5g / L preservative a second reagent comprising Including, the buffer is selected from one or a combination of tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, and trihydroxymethylmethane buffer; The buffer solution of the first reagent and the buffer solution of the second reagent are the same or different; The concentration of the buffer solution is 10 mmol / L to 500 mmol / L. The pH of the buffer is 6.5 to 8.0; the stabilizing agent is selected from one or a combination of bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; the surfactant is selected from one or a combination of Brij 23, Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, coconut fatty acid diethanolamide, and AEO7; the preservative is selected from one or a combination of azide, MIT, PC biological preservative, and thimerosal; the azide is selected from the group consisting of sodium azide, lithium azide, and PC-300; The substrate comprises glucose-6-phosphate and β-nicotinamide adenine dinucleotide; The detection kit according to claim 13.

17. A buffer solution comprising 100 mM to 300 mM of 5mM to 25mM substrate, 0.01μg / L to 1mg / L of anti-hapten antibodies, 100 mM to 300 mM NaCl, 1g / L to 5g / L of stabilizer, 1 g / L to 5 g / L of surfactant, and 1g / L to 5g / L preservative A first reagent comprising: 100 mM to 300 mM buffer, 8. The conjugate according to claim 6 or 7, at 0.05 μg / L to 0.5 mg / L. 1g / L to 5g / L of stabilizer, 1 g / L to 5 g / L of surfactant, and 1g / L to 5g / L preservative a second reagent comprising The detection kit of claim 16, comprising:

18. 1) preparing a glucose-6-phosphate dehydrogenase mutant according to claim 1; 2) preparing a hapten; 3) conjugating the glucose-6-phosphate dehydrogenase mutant with a hapten in a molar ratio of 1:3; Including, A method for preparing a conjugate, wherein step 1) and step 2) are performed in parallel or interchangeably successively.

19. The method of claim 18, wherein the hapten is selected from the group consisting of a small molecule drug, an antibiotic, a hormone, a metabolite, an amino acid, a polysaccharide, a lipid, and a short peptide; 19. The method of claim 18, wherein the hapten has a molecular weight of from 100 Da to 4000 Da.

20. The method of claim 19, wherein the hapten has a molecular weight of 200 Da to 1500 Da.

21. 1) preparing a glucose-6-phosphate dehydrogenase mutant according to claim 1; 2) providing a hapten or a derivative thereof; 3) contacting the glucose-6-phosphate dehydrogenase mutant with a hapten or a derivative thereof at 18° C. to 28° C. for 1 hour to 4 hours, thereby conjugating the hapten or a derivative thereof with the glucose-6-phosphate dehydrogenase mutant to obtain a conjugate; 4) purifying the conjugate; Including, Step 1) and step 2) are interchangeable or parallel; the buffer is selected from the group consisting of PBS, Tris, TAPS, and TAPSO; The pH of the buffer is 6.0 to 8.0; The aprotic solvent is selected from one or a combination of acetonitrile, dimethylformamide, and dimethylsulfoxide; A method for preparing the conjugate of claim 6 or 7.

22. The method of claim 20, wherein the glucose-6-phosphate dehydrogenase mutant contains one or more free sulfhydryl groups prior to step 3), contacting a glucose-6-phosphate dehydrogenase mutant with a hapten or a derivative thereof in a molar ratio of 1:n; n is 1 to 200; The method of claim 21.

23. The method of claim 22, wherein n is 30 to 120.

Images