CN111678874A - 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent - Google Patents

6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent Download PDF

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CN111678874A
CN111678874A CN202010013174.7A CN202010013174A CN111678874A CN 111678874 A CN111678874 A CN 111678874A CN 202010013174 A CN202010013174 A CN 202010013174A CN 111678874 A CN111678874 A CN 111678874A
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CN111678874B (en
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赵蕾
张启飞
王贵利
龚俊
刘希
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Beijing Strong Biotechnologies Inc
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a cyclosporine 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-glucose phosphate dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent
Priority of application No. 201910017764.4 filed on 1/9/2019 and 201910423122.4 "mutant glucose-6-phosphate dehydrogenase and use thereof in the preparation of test agents" filed on 5/21/2019, which are incorporated herein by reference.
Technical Field
The application relates to the field of biological detection, in particular to mutant enzyme 6-phosphoglucose dehydrogenase (G6 PDH for short) and application thereof in a cyclosporine 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.
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.
Cyclosporine A (Cyclosporine A, CsA) has the following structural formula:
Figure BDA0002357879600000021
cyclosporin A is a lipid-soluble cyclic peptide consisting of a variety of amino acids secreted by fungi (Trichoderma polyspora or Trichoderma columbiatum). The CsA is used as an immunosuppressant with high selectivity and ultralow bone marrow toxicity, can selectively inhibit the proliferation and the release of T helper lymphocytes, and simultaneously has antifungal activity, and can effectively reduce the infection condition of patients. The cyclosporin A has important clinical application value in organ and tissue transplantation, blood diseases, ophthalmic diseases and other aspects, and can prevent rejection reaction caused by organ or tissue transplantation of allogeneic kidney, liver, heart, bone marrow and the like and prevent and treat graft-versus-host reaction caused by bone marrow transplantation in the aspect of organ and tissue transplantation; in the aspect of hematopathy, the medicine can be applied to diseases such as aplastic anemia, red blood cell aplastic anemia, myelodysplastic syndrome, autoimmune hemolytic anemia and the like; in the aspect of ophthalmic diseases, the eye drops are mainly used for diseases such as Behcet's syndrome, xerophthalmia, scleritis, allergic conjunctivitis and the like.
Cyclosporin a, a commonly used drug as a calcineurin inhibitor, has become one of the important choices for the treatment of many refractory renal diseases and organ transplantation, and is mainly distributed in human tissues other than the brain, and mainly distributed in red blood cells and plasma in blood. Cyclosporine A is mainly metabolized by liver, 15 metabolites are known, the elimination half-life period is about 10-27 hours, the metabolites are mainly excreted by bile through feces, and only 0.1% of urine is excreted in the form of original drug.
However, the medicament has narrow safety range and complicated metabolism influence factors, obvious individual difference exists in pharmacokinetics and pharmacodynamics, the correlation between the blood concentration and the dosage is poor, the difficulty is increased for clinical treatment, adverse reactions such as nephrotoxicity, hirsutism, gingival hyperplasia and the like are easy to generate, and the individual difference of the adverse reactions is serious.
For the reasons, cyclosporine A blood concentration monitoring is required in time in the treatment process, and the method is an effective mode for assisting clinical treatment, improving treatment effect and reducing toxicity risk.
The currently known detection methods of cyclosporine A mainly comprise: high Performance Liquid Chromatography (HPLC), luminescence immunity, enzyme-linked immunosorbent assay (ELISA), etc. The HPLC method needs complex sample pretreatment, has complex operation and long period and is expensive in cost; the reagent of the luminescence immunoassay method has high cost, is not suitable for the detection of conventional treatment drugs, and is not more beneficial to large-scale popularization.
The existing homogeneous enzyme immunoassay method and latex agglutination turbidimetry method are limited in application due to complex preparation process and large batch difference.
The prior art CN107782889A describes a cyclosporine a detection kit, which discloses a preparation method of a conjugate of glucose-6-phosphate dehydrogenase and cyclosporine a:
accurately weighing 240mg of cyclosporin A and 50mg of 4-benzoylbenzoic acid, putting into a quartz cuvette, adding 10mL of tert-butyl alcohol, and ultrasonically dissolving;
irradiating by ultraviolet light for 3 hours, fully reacting, and standing at room temperature to form lyophilized powder;
accurately weighing 4.5mg of 6-phosphoglucose dehydrogenase and dissolving in 0.5mL of 10mM PBS (pH 7.4), dissolving 15mg of the above powder (CsA-BBa) in 0.7mL of Dimethylformamide (DMF), and adding CsA-BBa dropwise into the PBS solution containing GDH at constant temperature of 25 deg.C under stirring;
adjusting the pH value of the solution to be 6.0-8.0, dropwise adding 120 mu L of 0.5% carbodiimide (EDC), and stirring overnight at 4 ℃;
purifying by G-25 gel chromatography column to obtain 6-phosphoglucose dehydrogenase-cyclosporine A conjugate, and storing at 2-8 deg.C.
However, the prior art methods rely on activation of a reactive group carried by the small molecule drug (cyclosporin A) itself, followed by reaction with an enzyme. In such a coupling method, a plurality of cyclosporins a are linked to the same glucose-hexametaphosphate dehydrogenase, and it is difficult to ensure consistency of coupling sites and orientation between the small molecule drug and the enzyme 1: 1, resulting in large batch-to-batch variation.
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 the preparation of a cyclosporine a assay 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, D375C, G426C.
According to some embodiments, there is provided a glucose-6-phosphate dehydrogenase mutant, the glucose-6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.
According to some embodiments, 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: x is coupled. In some embodiments, x is 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. In some specific embodiments, the 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., cyclosporin A) that do not themselves bear a coupling group (e.g., a group that reacts with a sulfhydryl group) can be engineered to bear a linker for covalent attachment to a sulfhydryl 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.
Haptens are exemplified by, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin, digoxigenin), mycophenolic acid, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolites cotinine of uretonidine, morphine, derivatives of uromonohydric phenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, cortisol, Urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfates, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 α hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1 α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, nalin, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane a2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.
In a particular embodiment, the hapten is cyclosporine a or a derivative thereof.
In particular embodiments, the hapten is a cyclosporin a derivative bearing a thiol-reactive group, such as a maleimide, bromoacetyl, vinyl sulfone, or aziridine.
In a particular embodiment, the hapten is a cyclosporin a derivative, as shown in formula I:
Figure BDA0002357879600000051
wherein CsA is
Figure BDA0002357879600000052
Formula II.
In some embodiments, m is an integer from 1 to 10, preferably from 1 to 3, e.g., 1, 2, 3.
In a specific embodiment, the cyclosporin a derivative is represented by formula III:
Figure BDA0002357879600000061
wherein CsA is
Figure BDA0002357879600000062
Formula II; .
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 cyclosporine a detection reagent.
According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a cyclosporine 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 the use of a conjugate of the present application in the preparation of a cyclosporine a detection device.
In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., a 96-well plate), such as a plate coated with a reagent according to the present application.
In particular embodiments, the detection device may be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with a reagent according to the present application.
According to some embodiments, there is provided a cyclosporine a detection kit comprising:
-a first reagent comprising a substrate, a buffer and a cyclosporine a antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;
-a second agent comprising a conjugate of the present application and a buffer;
-optionally, a calibrator comprising 10mM to 500mM buffer, 0ng/ml to 1500ng/ml cyclosporin a; and
-optionally, a quality control comprising 10mM to 500mM buffer, 20ng/ml to 1400ng/ml cyclosporin a.
According to one embodiment, there is provided a cyclosporine a detection kit comprising:
a first reagent comprising:
10mM to 500mM buffer solution,
5mM to 50mM substrate,
0.1 to 10 mug/ml cyclosporin A antibody,
0.05% to 0.5% w/v stabilizer,
0.05% to 0.5% w/v of a surfactant,
0.05% to 0.5% w/v preservative;
a second reagent comprising:
10mM to 500mM buffer solution,
0.01. mu.g/ml to 1. mu.g/ml of a conjugate according to the present application,
0.05% to 0.5% w/v stabilizer,
0.05% to 0.5% w/v of a surfactant,
0.05% to 0.5% w/v preservative;
in some embodiments, the buffer is selected from one or a combination of: TAPS, 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 is 10mmol/L to 500mmol/L, preferably 50 to 100 mM; the pH of the buffer is 7 to 8.4.
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: brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 80.
In some embodiments, the preservative is selected from one or a combination of: azide, MIT, biological preservative PC (e.g. PC300), 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 specific embodiments, the cyclosporine a antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, human.
In some specific embodiments, the cyclosporin a antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, antigen-binding fragments.
According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:
1) providing a cyclosporine a derivative 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, preferably in a buffer (which provides a reaction environment, such as, but not limited to, PBS, Tris, TAPS, TAPSO, buffer pH between 6.0 and 8.0);
3) contacting the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant at a molar ratio n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) at 18 ℃ to 28 ℃ to couple the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant 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, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein n is 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, 100, 200, 300, 400, 500, and ranges between any of the foregoing values; preferably n is 20 to 60.
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 one or more free sulfhydryl groups, thereby allowing a targeted reaction with cyclosporine a.
Wild-type glucose-6-phosphate dehydrogenase does not contain a free sulfhydryl group, and thus in some embodiments, glucose-6-phosphate dehydrogenase is genetically engineered to have a free sulfhydryl group by mutating an amino acid at a specific site (306, 375, or 426) to cysteine.
Drawings
FIG. 1.G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.
FIG. 2.G6PDH (D306C) amino acid sequence (SEQ ID No. 2).
FIG. 3.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).
FIG. 4.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).
Detailed Description
Examples
Example 1 Synthesis of Cyclosporin A derivatives
Figure BDA0002357879600000091
1. Synthesis of Compound 2
Cyclosporin A (100mg,0.08mmol) was added to a round-bottomed flask, dissolved in dry dichloromethane (5mL), and triethylamine (25mg, 0.25mmol) was added to the reaction system, and stirred until all was dissolved.
Oxalyl chloride (52.8mg, 0.42mmol) was added to the reaction system, stirred at room temperature, checked by TLC, reacted at room temperature (18-28 ℃, preferably 20 to 25 ℃) for about 1 hour, the starting material substantially disappeared, the solvent was removed under reduced pressure, and excess oxalyl chloride was taken up repeatedly (2-3 times) using DCM and used in the next step without purification.
2. Synthesis of cyclosporin A derivatives
Compound 2 and compound 3(10mg, 0.05mmol) were dissolved in dry dichloromethane (5mL), to which triethylamine (12.4mg, 0.12mmol) was added dropwise, stirred at room temperature (18-28 ℃, preferably 20 to 25 ℃) and checked by TLC. After completion of the reaction, the reaction was directly purified using preparative plates (MeOH/DCM ═ 1:20) to give a cyclosporin a derivative of formula III (45mg, 38% yield).
3. The structure of the product was confirmed by a conventional method. This example allows cyclosporin A to carry a group that can bind to an enzyme.
Example 2 coupling of Cyclosporin A derivatives to G6PDH molecules
First, the coupling method of the present application
The coupling was carried out according to the G6 PDH-cyclosporine a conjugate of the present application in the following manner: a thiol-reactive group (such as, but not limited to, a maleimide group) on a cyclosporine a derivative molecule is covalently bound to a thiol on a G6PDH molecule.
1. The cyclosporine A derivative prepared in example 1 was dissolved in N, N-dimethylformamide (10 mg/ml);
g6PDH solution: g6PDH (mutant of the present application or of the prior art) was dissolved in 0.2M phosphate buffer, pH 8.0(2.5mg/ml enzyme);
3. adding 300 ul glucose 6 phosphate dehydrogenase mutant solution into 50 ul cyclosporine A derivative solution;
4. the mixed solution is shaken well for 2 to 3 hours at room temperature (18 to 28 ℃, preferably 20 to 25 ℃) and treated by molecular sieve chromatography, and the product, namely the G6 PDH-cyclosporine A conjugate (with the concentration of 0.1mg/mL to 2.5mg/mL) is obtained.
Two, control coupling method
The G6 PDH-cyclosporine a conjugate was prepared as disclosed in the example with reference to CN 107782889A.
Example 3 preparation of the kit
A kit for detecting cyclosporine a is prepared comprising:
reagent R1, comprising:
tris buffer 100mM, pH7.4
20mM glucose-6-phosphate
20mM beta-nicotinamide adenine dinucleotide
Cyclosporin A antibody 1. mu.g/ml (commercially available antibody)
0.5% w/v bovine serum albumin
0.1%w/v Tween80
0.1%w/v PC300;
Reagent R2, comprising:
tris buffer 100mM, pH8.2
0.05. mu.g/ml G6 PDH-Cyclosporin A conjugate
0.5% w/v bovine serum albumin
0.1%w/v Tween 80
0.1%w/v PC300;
Sample extraction liquid: 50mM of zinc sulfate, 50mM of Tris buffer solution, 3000.1% w/v of PC and 50% w/v of methanol;
calibration products: whole blood matrix, and 0ng/ml, 50ng/ml, 300ng/ml, 600ng/ml, 900ng/ml, 1500ng/ml cyclosporin A (or added as needed);
quality control product: whole blood matrix, and 100ng/ml, 750ng/ml, 1300ng/ml cyclosporin A (or added as needed).
And assembling the reagents (optionally containing quality control products and calibration products) into a detection kit.
The calibrator, the quality control product and the sample to be tested are physiological samples, such as serum, plasma, whole blood and the like. Preferably, the calibrator, the quality control material and the sample to be tested are all whole blood matrixes, and a sample extraction liquid is required to be pretreated before the test.
Example of detection
The kit adopts the enzyme amplification immunoassay EMIT principle, namely that the free CsA in the sample and the enzyme-CsA conjugate competitively bind to the CsA antibody, the more the free CsA in the sample is, the more the bound antibody is, the more the free conjugate is, the free conjugate catalyzes β -nicotinamide adenine dinucleotide oxidized form (NAD)+) Converting into β -nicotinamide adenine dinucleotide reduced form (NADH), wherein the CsA concentration in the sample is in direct proportion to the generation amount of NADH, and calculating the CsA content through the change of 340nm absorbance value.
TABLE 1 parameters of fully automatic biochemical analyzer
Model type Hitachi 7180 parameter
Sample size 2.0μl
Reagent R1 150μl
Reagent R2 50μl
Read point 27-34 points
Detecting wavelength (main) 340nm
Detecting wavelength (vice) 410nm
Curve fitting method Spline
Calibration article 0.0、50.0、300.0、600.0、900.0、1500.0ng/ml
Sample(s) Samples to be tested, e.g. plasma, serum, whole blood, urine, etc
Test example 1 Performance of the kit of the present application
1. Calibration of absorbance
TABLE 2 calibrated absorbances
Figure BDA0002357879600000121
2. Precision experiment
And measuring high, medium and low quality control products and clinical samples by using the calibration curve established above.
TABLE 3 Total inaccuracy
Figure BDA0002357879600000122
3. Repeatability of
And (4) measuring the low, medium and high-value quality control products for 20 times respectively to perform a repeatability experiment. As shown in the table below, the sample tests were repeated 20 times with CV within 2.35%.
TABLE 4 repeatability of the kit
Figure BDA0002357879600000131
4. Recovering
TABLE 5 recovery
Figure BDA0002357879600000132
5. Linearity of the detection kit
TABLE 6 linearity
Figure BDA0002357879600000141
Accelerated stability of reagent at 6.37 deg.C
TABLE 7.37 ℃ accelerated stability of reagents
Figure BDA0002357879600000142
7. Drug interference experiments
The following interferents were selected to produce a systematic bias of less than 10% at a cyclosporin A concentration of 200-1000 ng/ml.
TABLE 8 drug interference test
Medicine Test concentration (μ g/mL)
Aprazol cabin 0.57
Digoxin 0.015
Carbamazepine 100
Gentamicin 150
Rapamycin 0.25
Tacrolimus 0.25
Salicylic acid 400
Theophylline 200
Valproic acid 600
Vancomycin 500
Immunoglobulins 8500
Isoproterenol hydrochloride 0.4
Phenobarbital 200
Aminopyralid 5.0
Salbutamol 0.15
Medicine for treating stomach disease 4
Nitroglycerin 3.5
Test example 2 antibody inhibition Rate
1. Detection principle of antibody inhibition rate
When the antibody is combined with the G6 PDH-cyclosporine A conjugate, the activity of G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing the conversion of NAD into NADH is reduced, and the difference between an experimental group with the antibody added and an experimental group without the antibody added is compared by detecting the change of NADH amount, wherein the difference is reflected in the inhibition capacity of the antibody on G6 PDH.
2. Reaction system
TABLE 9 preparation of reagents for detection of antibody inhibition
Figure BDA0002357879600000151
3. Results
And (3) comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance value of the G6 PDH-cyclosporine A conjugate to obtain the inhibition condition of the antibody on G6 PDH.
Compared with the conjugate prepared from the published mutation sites (A45C and K55C), the mutant of the application has obviously improved antibody inhibition rate which can reach more than 44% (G426C: 44%; D375C: 50%) and reach 63% (D306C). Whereas the inhibition of previously published mutation sites (e.g. a45C, K55C) is 40% to 43%.
While not being bound to a particular theory, it may be partially explained as: compared with the G6PDH mutant (A45C, K55C) in the prior art, the mutation site (i.e. the site for introducing free sulfydryl) in the enzyme mutant of the application is the site for coupling with 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 the greatest effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it cannot 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 obviously improved antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the cyclosporine A is prepared into the kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, specificity and the like.
Sequence listing
<110> Beijing Jiuqiang Biotechnology Ltd
<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of cyclosporine A detection reagent
<130>300001CG
<150>201910017764.4
<151>2019-01-09
<150>201910423122.4
<151>2019-05-21
<160>4
<170>SIPOSequenceListing 1.0
<210>1
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<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)
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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
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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
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Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg
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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
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195 200 205
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225 230 235 240
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245 250 255
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260 265 270
Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala
275 280 285
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290 295 300
Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu
305 310 315 320
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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
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405 410 415
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420 425 430
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Ala Trp Val Phe Lys Gly
485
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<221>VARIANT
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Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys
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Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys
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145 150 155 160
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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
LeuCys 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
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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 GlyPro 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
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<221>VARIANT
<222>(375)..(375)
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325 330 335
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340 345 350
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355 360 365
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370 375 380
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385 390 395 400
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405 410 415
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485
<210>4
<211>486
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Ala Trp Val Phe Lys Gly
485

Claims (10)

1. A conjugate of a glucose-6-phosphate dehydrogenase mutant and a hapten in a molar ratio of 1: 1 is formed by covalent coupling;
the hapten is cyclosporine A or a derivative thereof;
preferably, the cyclosporin a derivative is represented by formula I:
Figure FDA0002357879590000011
wherein the content of the first and second substances,
CsA is
Figure FDA0002357879590000012
Formula II;
m is an integer of 1 to 10, preferably 1 to 3;
preferably, the cyclosporin a derivative is represented by formula III:
Figure FDA0002357879590000013
2. the conjugate of claim 1, wherein:
the glucose-6-phosphate dehydrogenase mutant comprises 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 and SEQ ID No. 4.
3. A reagent comprising the conjugate of claim 1 or 2.
4. Use of a conjugate according to claim 1 or 2 in the preparation of a detection reagent, wherein:
the detection reagent is a detection reagent of cyclosporine A;
preferably, the detection reagent is selected from: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.
5. Use of a conjugate according to claim 1 or 2 for the preparation of a detection device:
the detection device is a detection device for cyclosporine A;
the detection device is selected from any one of the following forms: orifice plate, particle, chip and test paper.
6. A cyclosporine a detection kit comprising:
a first reagent comprising a substrate, an anti-cyclosporin A antibody, a buffer;
a second reagent comprising the conjugate of claim 1 or 2, a buffer;
optionally, a calibrator comprising 0ng/ml to 1500ng/ml cyclosporin a; and
optionally, a quality control product comprising 20ng/ml to 1400ng/ml cyclosporin A.
7. The cyclosporine a detection kit of claim 6, comprising:
a first reagent comprising:
10mM to 500mM, preferably 50mM to 100mM, buffer,
5mM to 50mM, preferably 10mM to 20mM, glucose-6-phosphate,
5mM to 50mM, preferably 10mM to 20mM, oxidized form beta-nicotinamide adenine dinucleotide,
0.1 to 10. mu.g/ml, preferably 1 to 5. mu.g/ml, of an anti-cyclosporin A antibody,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v, of a stabilizer,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v, of a surfactant,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v preservative;
a second reagent comprising:
10mM to 500mM, preferably 50mM to 100mM, buffer,
The conjugate of claim 1 or 2, in an amount of 0.01 to 10. mu.g/ml, preferably 0.05 to 1. mu.g/ml,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v, of a stabilizer,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v, of a surfactant,
0.05% to 0.5% w/v, preferably 0.1% to 0.5% w/v preservative;
the buffer is selected from one or a combination of the following: TAPS buffer solution, phosphate buffer solution, glycine buffer solution, Tris buffer solution, boric acid buffer solution, MOPS buffer solution and HEPES buffer solution;
the pH of the buffer is 7 to 8.4;
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: brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 80;
the preservative is selected from one or a combination of the following: azide, MIT, biological preservative PC, thimerosal;
preferably, the preservative is selected from: sodium azide, lithium azide and PC-300.
8. The cyclosporine a detection kit of claim 6, comprising:
a first reagent comprising:
tris buffer 100mM, pH7.4,
20mM glucose-6-phosphate,
20mM oxidized beta-nicotinamide adenine dinucleotide,
1 mu g/ml anti-cyclosporin A antibody,
0.5% w/v bovine serum albumin,
0.1%w/v Tween80、
0.1%w/v PC300;
A second reagent comprising:
100mM Tris buffer, pH8.2,
0.05 μ g/ml of the conjugate according to claim 1 or 2,
0.5% w/v bovine serum albumin,
0.1%w/v Tween80、
0.1%w/v PC300。
9. A method of preparing a conjugate comprising the steps of:
1) providing cyclosporine A or a derivative thereof;
2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2;
3) the glucose-6-phosphate dehydrogenase mutant is coupled with the cyclosporine A or the derivative thereof;
the cyclosporine A derivative is represented by formula I:
Figure FDA0002357879590000041
wherein the content of the first and second substances,
CsA is
Figure FDA0002357879590000042
Formula II;
m is an integer of 1 to 10, preferably 1 to 3.
10. A method of preparing a conjugate according to claim 9, comprising the steps of:
1) providing a cyclosporin a derivative, preferably in an aprotic solvent;
2) providing a glucose-6-phosphate dehydrogenase mutant as defined in claim 2, preferably in a buffer;
3) contacting the glucose-6-phosphate dehydrogenase mutant and the cyclosporin A derivative at 18 ℃ to 28 ℃, preferably 20 ℃ to 25 ℃, for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that the cyclosporin A derivative and the glucose-6-phosphate dehydrogenase mutant are coupled to obtain the conjugate;
4) optionally, purifying the conjugate, preferably desalting the conjugate;
step 1) and step 2) can be interchanged or in parallel;
the buffer is selected from: PBS, Tris, TAPS, TAPSO,
the pH of the buffer is 6.0 to 8.0;
the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide;
preferably, prior to step 3), said glucose-6-phosphate dehydrogenase mutant comprises a free thiol group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375, or 426;
preferably, the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1, n is 1 to 500, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500;
more preferably, n is 30 to 60.
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CN202310507880.0A Pending CN116754756A (en) 2019-01-09 2020-01-07 Methotrexate detection kit
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CN202310553479.0A Pending CN116699125A (en) 2019-01-09 2020-01-07 Use of conjugates in the preparation of detection reagents
CN202310702870.2A Pending CN116840462A (en) 2019-01-09 2020-01-08 Method for preparing conjugate
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CN202310725902.0A Pending CN116577494A (en) 2019-01-09 2020-01-08 Use of conjugates in the preparation of detection reagents
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CN202310217235.5A Pending CN116359146A (en) 2019-01-09 2019-12-26 Method for preparing conjugate
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CN201911372147.2A Active CN112285037B (en) 2019-01-09 2019-12-27 6-phosphoglucose dehydrogenase mutant and application thereof in preparing detection reagent
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CN202310811498.9A Pending CN116698772A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202310811212.7A Pending CN116735512A (en) 2019-01-09 2019-12-27 Use of conjugates in the preparation of detection reagents
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
CN202211153004.4A Pending CN115808398A (en) 2019-01-09 2019-12-27 Method for preparing conjugate
CN202310726493.6A Pending CN116773795A (en) 2019-01-09 2019-12-31 Preparation method of conjugate
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CN202311025752.9A Pending CN117074335A (en) 2019-01-09 2020-01-02 Use of conjugates in the preparation of detection reagents
CN202311025762.2A Pending CN117054643A (en) 2019-01-09 2020-01-02 Vancomycin detection kit
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CN202310810455.9A Pending CN116840467A (en) 2019-01-09 2020-01-03 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
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
CN202310452740.8A Pending CN116430056A (en) 2019-01-09 2020-01-06 Method for preparing conjugate
CN202310320729.6A Pending CN116297271A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of kits
CN202310452946.0A Pending CN116559425A (en) 2019-01-09 2020-01-06 Use of conjugates in the preparation of detection reagents
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
CN202310453290.4A Pending CN116338215A (en) 2019-01-09 2020-01-06 Tacrolimus detection kit
CN202310318754.0A Pending CN116124721A (en) 2019-01-09 2020-01-06 Gentamicin detection kit
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CN202310555230.3A Pending CN116718761A (en) 2019-01-09 2020-01-07 Cyclosporine A detection kit
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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
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
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CN202310702860.9A Pending CN116699122A (en) 2019-01-09 2020-01-08 Rapamycin detection kit

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115236216A (en) * 2022-06-07 2022-10-25 合肥和合医疗科技有限公司 Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110174363A (en) * 2019-01-09 2019-08-27 北京九强生物技术股份有限公司 Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent
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CN112114127A (en) * 2020-09-09 2020-12-22 武汉生之源生物科技股份有限公司 Glycocholic acid homogeneous enzyme immunoassay kit and preparation method and application thereof
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CN112574969A (en) * 2020-12-28 2021-03-30 郑州伊美诺生物技术有限公司 G6PDH mutant and application thereof
CN113567662A (en) * 2021-07-08 2021-10-29 重庆中元汇吉生物技术有限公司 Kit for determining glycocholic acid and preparation method thereof
CN113736744B (en) * 2021-10-14 2023-07-18 江南大学 Digitoxin monoclonal antibody hybridoma cell strain and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686253A (en) * 1990-11-20 1997-11-11 Behringwerke Ag Method of stabilizing enzyme conjugates
US6054303A (en) * 1990-11-20 2000-04-25 Dade Behring Marburg Gmbh Cyclosporin immunoassay
CN1319105A (en) * 1998-10-09 2001-10-24 伊索技术公司 Methods for produsction of antibodies to specific regions of cyclosporine and cyclosporine metabolites
CN101120012A (en) * 2004-12-17 2008-02-06 伊素制药公司 Metabolites of cyclosporin analogs
CN110174363A (en) * 2019-01-09 2019-08-27 北京九强生物技术股份有限公司 Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent

Family Cites Families (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2116301B1 (en) * 1970-12-07 1974-08-30 Brun Lab Sa Le
US4190496A (en) * 1971-05-14 1980-02-26 Syva Company Homogeneous enzyme assay for antibodies
US3997525A (en) * 1974-01-16 1976-12-14 Bio-Tec, Inc. Tetra-125 iodo-di-tyramine of digitalis derivative and process for making the same
DE2901218A1 (en) * 1979-01-13 1980-07-17 Byk Gulden Lomberg Chem Fab THEOPHYLLIN
JPS5618983A (en) * 1979-07-25 1981-02-23 Eisai Co Ltd Theophylline derivative and its preapration
US4262089A (en) * 1980-04-07 1981-04-14 Syva Company Theophylline antigens and antibodies
US4341866A (en) * 1980-06-02 1982-07-27 Syva Company Antienzyme termination in enzyme immunoassays
JPS57178159A (en) * 1981-04-27 1982-11-02 Banyu Pharmaceut Co Ltd Chemical reagent for detection of amicacin and its determination
US4608336A (en) * 1981-08-27 1986-08-26 Miles Laboratories, Inc. #3B theophylline immunoassay employing 9-theophylline reagents
US4469797A (en) * 1982-09-23 1984-09-04 Miles Laboratories, Inc. Digoxigenin immunogens, antibodies, labeled conjugates, and related derivatives
EP0119767B1 (en) * 1983-03-11 1990-11-22 FUJIREBIO KABUSHIKI KAISHA also trading as FUJIREBIO INC. Method of measuring ligands
IT1199088B (en) * 1984-03-09 1988-12-30 Miles Italiana SPECIFIC BOND TEST BY USING ANTI-G6PDH AS A MARKER
US4622294A (en) * 1985-02-08 1986-11-11 Kung Viola T Liposome immunoassay reagent and method
US5068198A (en) * 1986-03-26 1991-11-26 Syntex (U.S.A.) Inc. Liquid single reagent for assays involving confining gels
EP0399127A1 (en) * 1989-05-23 1990-11-28 Pharmacia ENI Diagnostics Inc. Homogeneous immunochemical method for determining haptens by means of ion selective electrodes
DE3919915A1 (en) * 1989-06-19 1990-12-20 Boehringer Mannheim Gmbh AMINOALKYLMALEIMIDES AND DERIVED HAPTEN AND ANTIGEN DERIVATIVES AND CONJUGATES WITH PEPTIDES OR PROTEINS
JPH0833394B2 (en) * 1990-10-03 1996-03-29 三洋化成工業株式会社 Method for producing enzyme-labeled hapten
CA2087397A1 (en) * 1992-01-22 1993-07-23 Kazuhisa Kubotsu Immunoassay and reagents used therefor
CA2156397C (en) * 1993-04-08 2007-05-15 Valerie Quesniaux Rapamycin assay
US6455288B1 (en) * 1993-04-08 2002-09-24 Dade Behring Marburg Gmbh Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases
US5747352A (en) * 1994-05-23 1998-05-05 Beckman Instruments, Inc. Reagents and methods for the rapid and quantitative assay of pharmacological agents
AUPP751398A0 (en) * 1998-12-04 1999-01-07 Commonwealth Scientific And Industrial Research Organisation Methotrexate derivatives
US7078495B1 (en) * 1999-08-03 2006-07-18 Dade Behring Inc. Monoclonal antibodies to tacrolimus and immunoassay methods for tacrolimus
US6653456B2 (en) * 2001-07-31 2003-11-25 Roche Diagnostics Corporation Site-specific aminoglycoside derivatives and their use in immunodiagnostic assays
US20050176080A1 (en) * 2004-02-10 2005-08-11 Vani Bodepudi Hapten, immunogens and derivatives of ascomycin useful for preparation of antibodies and immunoassays
US20060046273A1 (en) * 2004-08-27 2006-03-02 Lin-Zhi International Inc. Homogeneous enzyme immunoassay for oral fluid
US7189582B2 (en) * 2005-04-27 2007-03-13 Dade Behring Inc. Compositions and methods for detection of sirolimus
JP4746926B2 (en) * 2005-06-29 2011-08-10 シスメックス株式会社 Glucose-6-phosphate dehydrogenase-containing reagent and glucose-6-phosphate dehydrogenase stabilization method
CN101638640B (en) * 2009-09-07 2011-01-12 北京利德曼生化股份有限公司 Glucose-6-phosphoric acid dehydrogenase and nucleotide sequence, recombinant vector, recombinant host cell and kit thereof
JP2014503197A (en) * 2010-11-24 2014-02-13 ディーエイチ テクノロジーズ デベロップメント プライベート リミテッド High-throughput, sensitive detection of glucose-6-phosphate dehydrogenase
CN102565399B (en) * 2010-12-07 2015-06-03 北京望尔生物技术有限公司 Method for detecting hydrocortisone and special enzyme-linked immunosorbent assay kit thereof
CN102807618A (en) * 2011-08-10 2012-12-05 重庆金域医学检验所有限公司 Preparation method of phenytoin homogeneous enzyme immunoassay kit and phenytoin polyclonal antibodies
JP5896375B2 (en) * 2011-09-09 2016-03-30 池田食研株式会社 Modified glucose dehydrogenase gene
CN102424829B (en) * 2011-10-26 2013-10-16 苏州汉酶生物技术有限公司 Method for synthesizing temsirolimus through enzyme catalysis
US8771964B2 (en) * 2012-02-02 2014-07-08 Siemens Healthcare Diagnostics Inc. Compositions and methods for detection of methadone metabolite
CN104619350A (en) * 2012-06-14 2015-05-13 Ambrx公司 Anti-psma antibodies conjugated to nuclear receptor ligand polypeptides
CN103242446A (en) * 2012-07-25 2013-08-14 苏州博源医疗科技有限公司 Theophylline immunogen and preparation method and application thereof
CN102757391B (en) * 2012-08-01 2015-08-26 苏州博源医疗科技有限公司 A kind of Phenobarbital derivatives and its preparation method and application
EP3083657B1 (en) * 2013-12-17 2022-01-26 Siemens Healthcare Diagnostics Inc. Preparation of multi-hapten mutant g6pdh conjugates and their use for detection of multiple analytes
CN104016923B (en) * 2014-01-08 2016-08-31 南开大学 Phenytoin derivant and its production and use
CN103739703B (en) * 2014-02-11 2015-07-15 苏州博源医疗科技有限公司 Glycocholic acid immunogen, anti-glycocholic acid specific antibody and detection reagent
CN103760348B (en) * 2014-02-11 2015-03-11 苏州博源医疗科技有限公司 Glycocholic acid immunodetection reagent and preparing method and detecting method thereof
JP6398295B2 (en) * 2014-04-30 2018-10-03 ニプロ株式会社 Mutant glucose-6-phosphate dehydrogenase
CN104447745B (en) * 2014-11-06 2016-03-30 济南金域医学检验中心有限公司 A kind of theophylline homogeneous enzyme immunoassay detects tests test kit and preparation method thereof
CN104569373B (en) * 2015-01-27 2016-08-17 苏州博源医疗科技有限公司 A kind of methotrexate homogeneous enzyme immunoassay detectable and preparation thereof and detection method
US10330683B2 (en) * 2015-02-04 2019-06-25 Genentech, Inc. Mutant smoothened and methods of using the same
CN105131105A (en) * 2015-07-27 2015-12-09 苏州博源医疗科技有限公司 Cortisol immunogen, derivative, antibody, detection reagent and preparation method
CN106565809B (en) * 2016-07-08 2018-05-01 北京九强生物技术股份有限公司 A kind of enzyme donor conjugate of beta galactosidase and its purposes in glycocholic acid detection
CN106226512B (en) * 2016-07-29 2018-10-16 胡清 A kind of detection method of kit, the preparation method of kit and the peripheral blood glycocholic acid realized using the kit
CN106190996B (en) * 2016-08-30 2019-05-21 美康生物科技股份有限公司 A kind of G 6 PD mutant
CN109797143B (en) * 2016-09-22 2021-02-12 北京九强生物技术股份有限公司 Reagent containing escherichia coli beta galactosidase receptor
CN106872681B (en) * 2017-01-23 2019-11-19 四川精卫食品检测科技有限公司 Amikacin and the two-in-one quick detection enzyme linked immunological kit of kanamycins and its application
CN108593905A (en) * 2017-12-22 2018-09-28 太原瑞盛生物科技有限公司 A kind of digoxin immune detection reagent and its preparation and detection method
CN108586562B (en) * 2018-05-08 2019-09-06 苏州博源医疗科技有限公司 A kind of cortex 01 derivatives and the preparation method and application thereof
CN108717117A (en) * 2018-05-23 2018-10-30 太原瑞盛生物科技有限公司 A kind of vancomycin immunologic function test reagent and its preparation and detection method
CN109111494A (en) * 2018-08-30 2019-01-01 苏州博源医疗科技有限公司 Derivatives of estradiol, immunogene, antibody, enzyme mark conjugate, detection reagent and preparation method thereof
CN112574969A (en) * 2020-12-28 2021-03-30 郑州伊美诺生物技术有限公司 G6PDH mutant and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5686253A (en) * 1990-11-20 1997-11-11 Behringwerke Ag Method of stabilizing enzyme conjugates
US6054303A (en) * 1990-11-20 2000-04-25 Dade Behring Marburg Gmbh Cyclosporin immunoassay
CN1319105A (en) * 1998-10-09 2001-10-24 伊索技术公司 Methods for produsction of antibodies to specific regions of cyclosporine and cyclosporine metabolites
CN101120012A (en) * 2004-12-17 2008-02-06 伊素制药公司 Metabolites of cyclosporin analogs
CN110174363A (en) * 2019-01-09 2019-08-27 北京九强生物技术股份有限公司 Glucose-6-phosphate dehydrogenase mutant and its purposes in preparation detection reagent

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115236216A (en) * 2022-06-07 2022-10-25 合肥和合医疗科技有限公司 Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof
CN115236216B (en) * 2022-06-07 2024-03-01 合肥和合医疗科技有限公司 Kit for detecting immunosuppressant in whole blood by high performance liquid chromatography tandem mass spectrometry, preparation method and detection method thereof

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