CN112285037A - 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent - Google Patents

6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent Download PDF

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CN112285037A
CN112285037A CN201911372147.2A CN201911372147A CN112285037A CN 112285037 A CN112285037 A CN 112285037A CN 201911372147 A CN201911372147 A CN 201911372147A CN 112285037 A CN112285037 A CN 112285037A
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龚俊
祁金祥
肖兰萍
张启飞
封建新
王贵利
刘希
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Beijing Strong Biotechnologies Inc
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Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a detection reagent. Specifically, the glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations selected from the group consisting of: D306C, D375C, G426C. The 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 accurate quantification, and is suitable for high-throughput detection.

Description

6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
The present application claims priority from 201910017764.4 filed on day 1/9 in 2019 and 201910423122.4 "glucose-6-phosphate dehydrogenase mutant and its use in the preparation of test agents" filed on day 21/5 in 2019, which are incorporated herein by reference.
Technical Field
The application relates to the field of biological detection, in particular to a multi-site mutant enzyme, namely 6-phosphoglucose dehydrogenase (G6 PDH for short) and application thereof in a detection kit.
Background
Haptens, some small molecular substances (molecular weight less than 4000Da), alone cannot induce an immune response, i.e. are not immunogenic, but can acquire immunogenicity when crosslinked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysine, and induce an immune response. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.
The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically bound to a protein molecule (carrier), it will acquire new immunogenicity and will stimulate the production of corresponding antibodies in animals. Haptens, once bound to a protein, constitute an antigenic cluster of the protein. Some chemical active group substances (such as penicillin, sulfanilamide, etc.) with molecular weight smaller than that of general hapten and specific structure are called simple hapten.
Small molecule antigens or haptens lack two or more sites that can be used in sandwich assays, and therefore cannot be measured using the double antibody sandwich assay, and often use a competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete to bind with the solid-phase antibody. The more the amount of the antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color develops. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.
Glycocholic acid (CG), which is a specific example of a hapten, is a conjugated cholic acid formed by binding cholic acid and glycine, and is one of main components of bile acid. Cholesterol undergoes a series of complex enzyme-catalyzed reactions in liver cells to form primary bile acid, which comprises Cholic Acid (CA) and chenodeoxycholic acid (CDCA), wherein the steroid nucleus of the cholic acid has three hydroxyl groups (C3, C7 and C12), and the hydroxyl group at the tail end of a side chain is combined with glycine by a peptide bond to form glycocholic acid (figure 1).
Glycocholic acid is synthesized by liver cells, is discharged into a gall bladder through a bile capillary and a bile duct, enters duodenum along with bile, and helps digestion and absorption of fat in food. 95% bile acid is reabsorbed in ileum and colon, returns to liver through portal vein, is absorbed and reused by liver cells, and the reabsorbed glycocholic acid enters liver-intestine circulation again, and through the mechanism, the organism can fully utilize glycocholic acid.
Normally, the content of cholic acid in peripheral blood is extremely low, and the content of glycocholic acid in peripheral blood is very low in normal people regardless of fasting or postprandial conditions. When human liver cells are damaged or bile is deposited, metabolism and circulation disorder of glycocholic acid can be caused, so that the capacity of the liver cells for absorbing glycocholic acid is reduced, the content of glycocholic acid in blood is increased, and the glycocholic acid value is related to the severity of liver cell damage and bile acid metabolic disturbance.
The determination of the content of glycocholic acid in serum is one of the sensitive indexes for evaluating the function of liver cells and the circulation function of liver and gall series substances. The glycocholic acid is more sensitive to liver function tests than conventional liver function tests such as ALT, AST, Total Bilirubin (TBIL), alkaline phosphatase (ALP), glutamyltranspeptidase (GGT), serum Albumin (ALB) and the like. Therefore, glycocholic acid can be used as a better detection index in liver function detection such as chronic hepatitis, acute hepatitis, liver cirrhosis, liver cancer, obstructive liver disease, liver and intestine circulatory disturbance, bile duct and gallbladder excretion dysfunction.
The currently known glycocholic acid detection methods mainly include: radioimmunoassay, enzyme-linked immunosorbent assay, chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography, mass spectrometry, etc. However, these detection methods all have many defects, such as radioactive contamination of radioimmunoassay isotope, short validity period, inconvenient operation and the like, and the enzyme-linked immunosorbent assay is relatively complicated in operation, takes a long time and is not suitable for clinical use. Although the chemiluminescence has good sensitivity, the chemiluminescence needs special equipment, and the cost of the chemiluminescence is high, so that the chemiluminescence is not beneficial to popularization. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex enhanced immunoturbidimetry are mainly used for detection.
Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, an enzyme-labeled antigen (such as G6PDH-CG) and a non-labeled antigen (CG) compete to be combined with a quantitative antibody (CG antibody), when the antibody is more combined with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH is generated by catalyzing a substrate NAD +, and the change of absorbance of NADH is detected under the wavelength of 340nm, so that the content of CG in the liquid can be calculated.
Disclosure of Invention
In view of the need in the art, the present application provides a novel glucose-6-phosphate dehydrogenase mutant and its use in preparing a glycocholic acid detection kit.
According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the previously published mutant of glucose-6-phosphate dehydrogenase of patent US006090567A (halogenated immunological systems using mutant glucose-6-phosphate dehydrogenes), the glucose-6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: D306C, G426C, D375C.
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, there is provided a polynucleotide encoding a glucose-6-phosphate dehydrogenase mutant of the present application.
According to some embodiments, there is provided an expression vector comprising a polynucleotide of the present application.
According to some embodiments, there is provided a host cell 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 of a glucose-6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1: n is coupled.
In some embodiments, n is 1 to 50, 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.
In some specific embodiments, the glucose-6-phosphate dehydrogenase mutant of the present application is preferably present in a molar ratio of 1: 1.
In some specific embodiments, the hapten has a molecular weight of from 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.
According to the present application, the skilled person will understand that "hapten" also comprises forms of its derivatives. To facilitate conjugation to glucose-6-phosphate dehydrogenase, haptens (e.g., CG) that do not themselves carry a coupling group (e.g., a group that reacts with a thiol group) can be engineered to carry a linker for covalent binding to the thiol group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.
The hapten is selected from: small molecule drugs (e.g. antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids, short peptides (molecular weight less than 4000kDa, or amino acids no longer than 50 residues in length).
Haptens are exemplified by, but not limited to: vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides, mycophenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites of uronicotin, cotinine, uromorphine, derivatives of uromonophenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormones, prolactin, placental prolactin, growth hormones, follicle stimulating hormones, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, retro-triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxysteroids, urinary 17-ketosteroids, mycophenolic acid, mycophenols, mycophenol, Dehydroepiandrosterone and sulfate, aldosterone, urovanillyl mandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 alpha hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, peptide C, gastrin, plasma prostaglandin, plasma 6-one prostaglandin F1 alpha, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.
In a particular embodiment, the hapten is glycocholic acid or a derivative thereof. Although glycocholic acid is taken as a specific example, it will be understood by the skilled person that the technical effect of the present application is independent of the particular type of hapten, which is applicable to any hapten which can be immunologically detected by means of competition methods.
In particular embodiments, the hapten is a glycocholic acid derivative, which carries a thiol-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine. In a particular embodiment, the hapten is a glycocholic acid derivative, represented by formula I:
Figure BDA0002339951490000051
according to some embodiments, there is provided a reagent 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 test agent.
According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a detection reagent.
In specific embodiments, the detection reagent is selected from the group consisting of: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.
In a specific embodiment, the detection reagent is preferably a reagent for detection based on a competition method.
According to some embodiments, there is provided a glycocholic acid detection kit comprising:
-a first reagent comprising a substrate and a glycocholic acid antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;
-a second agent comprising a conjugate of the present application;
-optionally, a calibrator comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid; and
-optionally, a quality control comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid.
According to one embodiment, there is provided a glycocholic acid detection kit comprising:
a first reagent comprising:
10mM to 500mM buffer solution,
5mM to 25mM substrate,
0.1mg/L to 1mg/L of glycocholic acid antibody,
10mM to 300mM NaCl,
0.1 to 5g/L stabilizer,
0.1g/L to 5g/L of surfactant,
0.1g/L to 5g/L preservative;
a second reagent comprising:
10mM to 500mM buffer solution,
The conjugate of claim 5,
0.1 to 5g/L 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: tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer; the concentration of the buffer solution is 10mmol/L to 500mmol/L, preferably 100 mM; the pH of the buffer is 6.5 to 7.5, preferably 7.2 or 7.0.
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, polyethylene glycol 8000; bovine serum albumin is preferred.
In some embodiments, the surfactant is selected from one or a combination of: brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.
In some embodiments, the preservative is selected from one or a combination of: azide, MIT, PC-300, thimerosal; the azide is selected from: sodium azide and lithium azide.
In some embodiments, the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.
In some embodiments, the concentration of the buffer is 100 mM.
In some embodiments, the substrate concentration of the reaction catalyzed by the G6PDH enzyme is 5 mM.
In some embodiments, the concentration of the glycocholic acid antibody is 0.1 mg/L.
In some embodiments, the concentration of NaCl is 300 mM.
In some embodiments, the concentration of the stabilizer is 0.5 g/L.
In some embodiments, the concentration of surfactant is 0.1 g/L.
In some embodiments, the concentration of the preservative is 1 g/L.
According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:
1) providing a hapten or a derivative thereof according to the present application, in particular in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethylsulfoxide);
2) providing a glucose-6-phosphate dehydrogenase mutant of the present application, preferably in a buffer (which provides a reaction environment, such as, but not limited to, PBS, Tris, TAPS, TAPSO, the buffer pH being 6.0 to 8.0);
3) (ii) contacting the glucose-6-phosphate dehydrogenase mutant and the hapten or derivative thereof in a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow the hapten or derivative thereof and the glucose-6-phosphate dehydrogenase mutant to be conjugated to obtain the seed conjugate;
4) the conjugate is optionally subjected to purification, such as desalting treatment or the like, as required.
In some embodiments, n is 1 to 50, 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.
In some specific embodiments, steps 1) and 2) are interchangeable or concurrent.
In some specific embodiments, the glucose-6-phosphate dehydrogenase, prior to conjugation, comprises a free sulfhydryl group, thereby allowing for 1: 1.
Drawings
FIG. 1 shows the structure of glycocholic acid.
FIG. 2 shows the structure of glycocholic acid derivatives.
FIG. 3A. G6PDH (wild-type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides of Leuconostoc.
FIG. 3B. G6PDH (D306C) amino acid sequence (SEQ ID No. 2).
FIG. 3C.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).
FIG. 3D.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).
Detailed Description
Examples
EXAMPLE 1 Synthesis of Glycocholic acid derivatives
To a dry, clean 25mL two-necked bottle was added glycocholic acid (1.0eq), maleimidoethylamine (1.0g, 1.0eq), triethylamine (3.0 eq);
then dimethylformamide (5mL) is added and stirred to be completely dissolved, dichloroethane (1.25eq) is added and stirred for 2h at 25 ℃;
monitoring by HPLC until the reaction is finished;
the reaction mixture was added to water (25mL), and ethyl acetate (20 mL. times.3) was added to conduct extraction;
the organic phases were combined and anhydrous Na2SO4Drying, concentrating under reduced pressure, and purifying the resulting oil by column chromatography to give 1.04g of a milky white powdery solid in a yield of 45%, M +: 602.72.
the effect of this example is to make CG with a group that can bind to the enzyme, and the technical effect of the present application is independent of the particular hapten derivative.
Example 2 coupling of Glycocholic acid derivatives to G6PDH molecules
The coupling was performed according to the G6 PDH-glycocholic acid conjugate of the present application in the following manner: the thiol-reactive group (e.g., maleimide group) on the glycocholic acid derivative molecule is covalently bound to a thiol group on the G6PDH molecule.
1. Solution preparation:
glycocholic acid derivative solution: the glycocholic acid derivative prepared in example 1 at 10mg/ml was dissolved in DMF;
g6PDH solution: 6.7mg/mL G6PDH (mutant or control mutant of the present application), PB 100mmol, NaCl 100mmol, pH 8.0;
coupling solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH 7.2;
desalting solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH 7.2.
2. Coupling operation: 1.6ml of G6PDH solution, 6ml of coupling solution and 0.40ml of glycocholic acid derivative solution were reacted at room temperature (20 to 25 ℃) for 4 hours.
3. And (3) oscillating the reaction system at room temperature for 4h, eluting with the desalting solution by using a desalting column, and collecting protein peaks to obtain a product, namely the G6 PDH-glycocholic acid conjugate.
Example 3 preparation of the kit
A kit for detecting glycocholic acid was prepared, which comprises:
reagent R1, comprising:
100mM PB buffer, pH 7.2
15mM glucose 6-phosphate
15mM beta-nicotinamide adenine dinucleotide
0.1mg/L glycocholic acid antibody
200mM NaCl
0.5g/L bovine serum albumin
0.1g/L Tween20
1g/L sodium azide;
reagent R2, comprising:
100mM PB buffer, pH 7.2
0.1mg/L G6PDH-CG conjugate
0.5g/L bovine serum albumin
0.1g/L Tween 20
1g/L sodium azide;
calibration products: 100mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 40mg/L glycocholic acid (or added as needed);
quality control product: 100mM PB buffer, pH 7.2, and 1.5, 8.0, 25, 35mg/L glycocholic acid (or added as needed).
Example of detection
Reaction time: 10min, wherein the incubation time was 4.7min, after 1min of incubation after addition of reagent R2, the read absorbance a1 was determined, after 1min of incubation, the read absorbance a2 was determined and Δ a ═ a (a2-a1)/min was calculated. The content of glycocholic acid in the sample was calculated by a calibration curve: CG is sample tube absorbance calibrator concentration/calibrator absorbance.
The glycocholic acid detection kit prepared in example 3 is subjected to performance detection, and the main detection performances are total inaccuracy, repeatability, recovery, linearity, specificity and the like.
TABLE 1 parameters of fully automatic biochemical analyzer
Detecting machine type Hitachi 7180
analysis/time/Point 2 point speed/10 min/20-24 points
R1/R2/S 120:40:9
Wavelength (auxiliary/main) 405/340
Type of reaction Incremental increase
Calibration type Spine type
Calibration point 6
Concentration of calibrator 0/2.50/5.00/10.00/20.00/40.00
Detection example 1 Glycocholic acid detection kit calibration Absorbance
TABLE 2 Glycocholic acid detection kit calibration absorbance
Figure BDA0002339951490000101
Detection example 2 Total inaccuracy degree of Glycocholic acid detection kit
TABLE 3 Total inaccuracy
Figure BDA0002339951490000102
Detection example 3 Glycocholic acid detection kit repeatability
TABLE 4 repeatability
Figure BDA0002339951490000111
Detection example 4 Glycocholic acid detection kit recovery
TABLE 5 recovery
Figure BDA0002339951490000112
Figure BDA0002339951490000121
Detection example 5 Glycocholic acid detection kit Linearity
TABLE 6 linearity
Figure BDA0002339951490000122
Figure BDA0002339951490000131
Detection example 6 Glycocholic acid detection kit anti-specificity
TABLE 7 specificity
Interferent (40. mu.g/ml) Reagent of the present application (D306C) Control reagent (A45C mutant)
Glycodeoxycholic acid 18.61％ 30.20％
Glycine chenodeoxycholic acid 1.63％ 37.91％
Chenodeoxycholic acid 0.61％ 16.28％
Ursodeoxycholic acid -0.42％ 6.55％
Cholesterol acid sodium salt 61.21％ 61.90％
Deoxycholic acid sodium salt 4.22％ 11.74％
The reagent of the present application has little to no cross-reaction with the structural analog of glycocholic acid.
Test example 7 antibody inhibition Rate
1. Detection principle of antibody inhibition rate
When the antibody is combined with the G6PDH-CG conjugate, the activity of 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 experimental group in which the antibody is added and an experimental group in which the antibody is not added is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.
2. Reaction system:
TABLE 8 preparation of reagents for measuring antibody inhibition
Figure BDA0002339951490000141
TABLE 9 antibody inhibition Rate testing of on-machine parameters
Detecting machine type Hitachi 7180
analysis/time/Point 2 point speed/10 min/20-24 points
R1/S 120:20
Wavelength (auxiliary/main) 405/340
Type of reaction Incremental increase
3. As a result:
and comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance values of the G6PDH-CG conjugate to obtain the inhibition situation of the antibody on G6 PDH.
The antibody inhibition rate is the change in absorbance of G6PDH-CG with antibody/the change in absorbance of G6PDH-CG without antibody.
Compared with published mutation sites, the mutant of the application has obviously improved antibody inhibition rate which can reach more than 30 percent and can reach 50 percent. Whereas the inhibition rate of the mutation sites (such as A45C and K55C) which are commonly used before is only about 20 percent or even lower.
TABLE 10 antibody inhibition of different G6PDH mutants
Figure BDA0002339951490000142
Figure BDA0002339951490000151
While not being bound to a particular theory, it may be partially explained as: compared with the G6PDH mutant in the prior art, the mutant (D306C, D375C, G426C) of the enzyme of the application has a mutation site (i.e., a site for introducing a free thiol) at a position for coupling with a hapten (such as hormone, small molecule drug and the like). When the hapten binds to a hapten-specific antibody at this position, the steric hindrance formed has minimal effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it does not substantially affect the steric folding of the molecule. Therefore, the position of this mutation site is very important, and needs to be compatible with the activity of G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.
The enzyme mutant has obvious advantage in calibration absorbance due to obvious improvement of the antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the hapten is prepared into the kit, the kit has obvious performance improvement in the aspects of repeatability, total inaccuracy, linearity, specificity and the like due to the improvement of a calibration curve.
Sequence listing
<110> Beijing Jiuqiang Biotechnology Ltd
<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of detection reagent
<130> 390313CG-390017
<150> 201910017764.4
<151> 2019-01-09
<150> 201910423122.4
<151> 2019-05-21
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 486
<212> PRT
<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)
<400> 1
Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly
1 5 10 15
Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
20 25 30
Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln
35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe
65 70 75 80
Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu
85 90 95
Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn
100 105 110
Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala
115 120 125
Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
130 135 140
Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu
145 150 155 160
Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg
165 170 175
Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu
180 185 190
Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile
195 200 205
Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg
210 215 220
Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn
225 230 235 240
His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser
245 250 255
Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala
260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu
290 295 300
Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg
325 330 335
Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe
340 345 350
Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala
355 360 365
Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu
370 375 380
Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu
385 390 395 400
Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr
405 410 415
Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala
420 425 430
Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser
435 440 445
Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly
450 455 460
Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp
465 470 475 480
Ala Trp Val Phe Lys Gly
485
<210> 2
<211> 486
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> VARIANT
<222> (306)..(306)
<223> G6PDH mutant, D at position 306 was replaced with C compared to wild type
<400> 2
Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly
1 5 10 15
Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
20 25 30
Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln
35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe
65 70 75 80
Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu
85 90 95
Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn
100 105 110
Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala
115 120 125
Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
130 135 140
Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu
145 150 155 160
Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg
165 170 175
Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu
180 185 190
Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile
195 200 205
Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg
210 215 220
Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn
225 230 235 240
His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser
245 250 255
Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala
260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu
290 295 300
Leu Cys Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg
325 330 335
Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe
340 345 350
Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala
355 360 365
Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu
370 375 380
Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu
385 390 395 400
Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr
405 410 415
Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala
420 425 430
Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser
435 440 445
Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly
450 455 460
Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp
465 470 475 480
Ala Trp Val Phe Lys Gly
485
<210> 3
<211> 486
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> VARIANT
<222> (375)..(375)
<223> G6PDH mutant, D at position 375 was replaced with C compared to wild type
<400> 3
Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly
1 5 10 15
Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
20 25 30
Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln
35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe
65 70 75 80
Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu
85 90 95
Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn
100 105 110
Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala
115 120 125
Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
130 135 140
Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu
145 150 155 160
Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg
165 170 175
Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu
180 185 190
Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile
195 200 205
Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg
210 215 220
Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn
225 230 235 240
His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser
245 250 255
Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala
260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu
290 295 300
Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg
325 330 335
Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe
340 345 350
Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala
355 360 365
Val Leu Ser Ile Ile Ile Cys Pro Lys Gly Ala Ile Glu Leu Lys Leu
370 375 380
Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu
385 390 395 400
Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr
405 410 415
Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala
420 425 430
Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser
435 440 445
Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly
450 455 460
Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp
465 470 475 480
Ala Trp Val Phe Lys Gly
485
<210> 4
<211> 486
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> VARIANT
<222> (426)..(426)
<223> G6PDH mutant, G at position 426 was replaced with C compared to wild type
<400> 5
Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly
1 5 10 15
Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
20 25 30
Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln
35 40 45
Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
50 55 60
Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe
65 70 75 80
Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu
85 90 95
Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn
100 105 110
Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala
115 120 125
Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
130 135 140
Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu
145 150 155 160
Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg
165 170 175
Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu
180 185 190
Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile
195 200 205
Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg
210 215 220
Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn
225 230 235 240
His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser
245 250 255
Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala
260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu
290 295 300
Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg
325 330 335
Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe
340 345 350
Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala
355 360 365
Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu
370 375 380
Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu
385 390 395 400
Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr
405 410 415
Glu Arg Met Ile His Asp Thr Met Asn Cys Asp Gly Ser Asn Phe Ala
420 425 430
Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser
435 440 445
Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly
450 455 460
Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp
465 470 475 480
Ala Trp Val Phe Lys Gly
485

Claims (10)

1. A glucose-6-phosphate dehydrogenase mutant comprising a mutation selected from the group consisting of: D306C, D375C, G426C;
preferably, the glucose-6-phosphate dehydrogenase mutant is represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.
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.
5. A conjugate of the glucose-6-phosphate dehydrogenase mutant of claim 1 with a hapten in a molar ratio of 1: n is coupled;
n is 1 to 50, preferably n is 1;
preferably, the hapten is selected from the group consisting of: small molecule drugs, antibiotics, hormones, metabolites, polysaccharides, lipids, short peptides;
the hapten has a molecular weight of 100Da to 4000Da, preferably 200Da to 1500 Da;
more preferably, the hapten is glycocholic acid or a derivative thereof.
6. A reagent comprising the conjugate of claim 5.
7. Use of any one of the following in the preparation of a test agent:
the glucose-6-phosphate dehydrogenase mutant of claim 1, the conjugate of claim 5;
preferably, the detection reagent is a detection reagent for a hapten;
preferably, the detection reagent is selected from: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent;
preferably, the hapten is selected from the group consisting of: small molecule drugs, antibiotics, hormones, metabolites, polysaccharides, lipids, short peptides;
the hapten has a molecular weight of from 100Da to 4000Da, preferably from 200Da to 1500 Da.
8. A glycocholic acid detection kit, comprising:
a first reagent comprising a substrate, a glycocholic acid antibody, a buffer;
a second reagent comprising the conjugate of claim 5, a buffer;
optionally, a calibrator comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid; and
optionally, a quality control product comprising 10mM to 500mM buffer, 0mg/L to 40mg/L glycocholic acid.
9. The glycocholic acid detection kit of claim 8, comprising:
a first reagent comprising:
10mM to 500mM, preferably 100mM to 300mM, buffer,
5mM to 25mM substrate,
0.1mg/L to 1mg/L of glycocholic acid antibody,
10mM to 300mM, preferably 100mM to 300mM NaCl,
0.1 to 5g/L, preferably 1 to 5g/L, of a stabilizer,
0.1g/L to 5g/L, preferably 1g/L to 5g/L, of a surfactant,
0.1g/L to 5g/L, preferably 1g/L to 5g/L preservative;
a second reagent comprising:
10mM to 500mM, preferably 100mM to 300mM, buffer,
0.01mg/L to 1mg/L, preferably 0.05mg/L to 0.5mg/L of the conjugate of claim 5,
0.1 to 5g/L, preferably 1 to 5g/L, of a stabilizer,
0.1g/L to 5g/L, preferably 1g/L to 5g/L, of a surfactant,
0.1g/L to 5g/L, preferably 1g/L to 5g/L preservative;
the buffer is selected from one or a combination of the following: tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer;
the concentration of the buffer solution is 10mmol/L to 500 mmol/L;
the pH of the buffer is 6.5 to 7.5;
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;
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 Tween 20;
the preservative is selected from one or a combination of the following: azide, MIT, PC-300, thimerosal;
the azide is selected from: sodium azide and lithium azide;
the substrate comprises: 6-phosphoglucose, beta-nicotinamide adenine dinucleotide.
10. A method of preparing a conjugate comprising the steps of:
1) providing a glucose-6-phosphate dehydrogenase mutant of claim 1;
2) providing a hapten, preferably said hapten is selected from the group consisting of: small molecule drugs, antibiotics, hormones, metabolites, polysaccharides, lipids, short peptides; the hapten has a molecular weight of 100Da to 4000Da, preferably 200Da to 1500 Da;
3) the glucose-6-phosphate dehydrogenase mutant and the hapten are mixed according to a molar ratio of 1: n is coupled; n is 1 to 50, preferably n is 1;
more preferably, the hapten is glycocholic acid or a derivative thereof;
step 1) and step 2) may be in parallel or in an alternating order.
CN201911372147.2A 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent Active CN112285037B (en)

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CN201911365439.3A Active CN111239060B (en) 2019-01-09 2019-12-26 6-phosphoglucose dehydrogenase mutant and application thereof in preparing theophylline detection reagent
CN202310257027.8A Pending CN116144619A (en) 2019-01-09 2019-12-26 Theophylline detection kit
CN202310217235.5A Pending CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
CN201911372535.0A Active CN112285038B (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of digitoxin detection reagent
CN202310811212.7A Pending CN116735512A (en) 2019-01-09 2019-12-27 Use of conjugates in the preparation of detection reagents
CN202211153004.4A Pending CN115808398A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202211151405.6A Pending CN115791649A (en) 2019-01-09 2019-12-27 Glycocholic acid detection kit
CN202211151264.8A Pending CN116008201A (en) 2019-01-09 2019-12-27 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of detection reagent
CN202310811498.9A Pending CN116698772A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN201911372147.2A Active CN112285037B (en) 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
CN202310811210.8A Pending CN116626281A (en) 2019-01-09 2019-12-27 Digitoxin detection kit
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CN201911403882.5A Active CN111650135B (en) 2019-01-09 2019-12-31 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of phenytoin detection reagent
CN202310726493.6A Pending CN116773795A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
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CN202010004879.2A Active CN111487207B (en) 2019-01-09 2020-01-03 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of digoxin detection reagent
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CN202310318754.0A Pending CN116124721A (en) 2019-01-09 2020-01-06 Gentamicin detection kit
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CN202010009771.2A Active CN111504921B (en) 2019-01-09 2020-01-06 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of gentamicin detection reagent
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CN202310257027.8A Pending CN116144619A (en) 2019-01-09 2019-12-26 Theophylline detection kit
CN202310217235.5A Pending CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
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CN202310364997.8A Pending CN116298257A (en) 2019-01-09 2019-12-31 Phenytoin detection kit
CN201911404154.6A Active CN111504920B (en) 2019-01-09 2019-12-31 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent
CN201911403882.5A Active CN111650135B (en) 2019-01-09 2019-12-31 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of phenytoin detection reagent
CN202310726493.6A Pending CN116773795A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
CN202310364266.3A Pending CN116718764A (en) 2019-01-09 2019-12-31 Use of conjugate in preparation of phenytoin detection reagent
CN202310724066.4A Pending CN116773827A (en) 2019-01-09 2019-12-31 Use of conjugates in the preparation of detection reagents
CN202310726498.9A Pending CN116559472A (en) 2019-01-09 2019-12-31 Cortisol detection kit
CN202310365160.5A Pending CN116355873A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
CN202010000321.7A Active CN111487206B (en) 2019-01-09 2020-01-02 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of vancomycin detection reagent
CN202311025762.2A Pending CN117054643A (en) 2019-01-09 2020-01-02 Vancomycin detection kit
CN202311025752.9A Pending CN117074335A (en) 2019-01-09 2020-01-02 Use of conjugates in the preparation of detection reagents
CN202311025756.7A Pending CN117030640A (en) 2019-01-09 2020-01-02 Method for preparing conjugate
CN202010004879.2A Active CN111487207B (en) 2019-01-09 2020-01-03 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of digoxin detection reagent
CN202310811497.4A Pending CN116840468A (en) 2019-01-09 2020-01-03 Use of conjugates in the preparation of detection reagents
CN202310810455.9A Pending CN116840467A (en) 2019-01-09 2020-01-03 Method for preparing conjugate
CN202310810479.4A Pending CN116819060A (en) 2019-01-09 2020-01-03 Digoxin detection kit
CN202010009570.2A Active CN111537451B (en) 2019-01-09 2020-01-06 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of tacrolimus detection reagent
CN202310318754.0A Pending CN116124721A (en) 2019-01-09 2020-01-06 Gentamicin detection kit
CN202310453290.4A Pending CN116338215A (en) 2019-01-09 2020-01-06 Tacrolimus detection kit
CN202310320729.6A Pending CN116297271A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of kits
CN202010009771.2A Active CN111504921B (en) 2019-01-09 2020-01-06 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of gentamicin detection reagent
CN202310320137.4A Pending CN116148198A (en) 2019-01-09 2020-01-06 Preparation method of gentamicin detection reagent
CN202310452946.0A Pending CN116559425A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of detection reagents
CN202310452740.8A Pending CN116430056A (en) 2019-01-09 2020-01-06 Method for preparing conjugate
CN202010013174.7A Active CN111678874B (en) 2019-01-09 2020-01-07 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent
CN202310554774.8A Pending CN116679047A (en) 2019-01-09 2020-01-07 Method for preparing conjugate
CN202310507880.0A Pending CN116754756A (en) 2019-01-09 2020-01-07 Methotrexate detection kit
CN202010013644.XA Active CN111487208B (en) 2019-01-09 2020-01-07 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of methotrexate detection reagent
CN202310508217.2A Pending CN116298330A (en) 2019-01-09 2020-01-07 Use of conjugates in the preparation of detection reagents
CN202310508418.2A Pending CN116381253A (en) 2019-01-09 2020-01-07 Method for preparing conjugate
CN202310553479.0A Pending CN116699125A (en) 2019-01-09 2020-01-07 Use of conjugates in the preparation of detection reagents
CN202310555230.3A Pending CN116718761A (en) 2019-01-09 2020-01-07 Cyclosporine A detection kit
CN202310726230.5A Pending CN116577495A (en) 2019-01-09 2020-01-08 Method for preparing conjugate
CN202310702870.2A Pending CN116840462A (en) 2019-01-09 2020-01-08 Method for preparing conjugate
CN202010016535.3A Active CN111693473B (en) 2019-01-09 2020-01-08 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of rapamycin detection reagent
CN202010017376.9A Active CN111537452B (en) 2019-01-09 2020-01-08 Glucose 6-phosphate dehydrogenase mutant and application thereof in preparation of amikacin detection reagent
CN202310725902.0A Pending CN116577494A (en) 2019-01-09 2020-01-08 Use of conjugates in the preparation of detection reagents
CN202310702860.9A Pending CN116699122A (en) 2019-01-09 2020-01-08 Rapamycin detection kit
CN202310726069.1A Pending CN116754761A (en) 2019-01-09 2020-01-08 Amikacin detection kit
CN202310702858.1A Pending CN116859035A (en) 2019-01-09 2020-01-08 Use of conjugates in the preparation of detection reagents

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