Use of conjugates in the preparation of detection reagents

Abstract

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

Description

[0001] This application is a divisional application of the patent application "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Cyclosporine A Detection Reagent" with application number 2020100131747 filed on January 7, 2020. Technical Field

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

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

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot stimulate antibody production in humans or animals on its own. It is only immunoreactive, not immunogenic, and is also called an incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs are haptens. If a hapten is chemically combined with a protein molecule (carrier), it acquires new immunogenicity and can stimulate the production of corresponding antibodies in animals.

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

[0006] The structural formula of cyclosporine A (CsA) is shown below:

[0007]

[0008] Cyclosporin A is a fat-soluble cyclic peptide composed of multiple amino acids secreted by a fungus (Trichoderma polysporum or Trichoderma cylindrosporum). As an immunosuppressant with high selectivity and ultra-low myelotoxicity, CsA selectively inhibits the proliferation and release of T helper lymphocytes. It also exhibits antifungal activity, effectively reducing infection in patients. Cyclosporin A has important clinical applications in organ and tissue transplantation, hematological diseases, and ophthalmic diseases. In organ and tissue transplantation, it can prevent rejection reactions associated with allogeneic kidney, liver, heart, bone marrow, and other organ or tissue transplants, and can also prevent and treat graft-versus-host reactions associated with bone marrow transplantation. In hematological diseases, it can be used for diseases such as aplastic anemia, red blood cell aplasia, myelodysplastic syndrome, and autoimmune hemolytic anemia. In ophthalmic diseases, it is primarily used for diseases such as Behçet's syndrome, dry eye, scleritis, and allergic conjunctivitis.

[0009] Cyclosporine A, a commonly used calcineurin inhibitor, has become an important treatment option for many refractory kidney diseases and organ transplantation. It is primarily distributed in human tissues, excluding the brain, and in the bloodstream, primarily in red blood cells and plasma. Cyclosporine A is primarily metabolized by the liver, with 15 known metabolites and an elimination half-life of approximately 10-27 hours. Metabolites are primarily excreted in bile and feces, with only 0.1% excreted in urine as the original drug.

[0010] However, due to the narrow safety range of the drug and the complex metabolic factors, there are obvious individual differences in pharmacokinetics and pharmacodynamics, and the poor correlation between blood drug concentration and dosage, which increases the difficulty of clinical treatment and is prone to adverse reactions such as nephrotoxicity, hirsutism, gingival hyperplasia, and serious individual differences in adverse reactions.

[0011] For the above reasons, timely monitoring of cyclosporine A blood concentration is necessary during treatment, which is an effective way to assist clinical treatment, improve treatment effects, and reduce toxicity risks.

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

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

[0014] Prior art CN107782889A describes a cyclosporin A detection kit, which discloses a method for preparing a conjugate of 6-phosphate glucose dehydrogenase and cyclosporin A:

[0015] Accurately weigh 240 mg of cyclosporine A and 50 mg of 4-benzoylbenzoic acid, place them in a quartz cuvette, add 10 mL of tert-butanol, and sonicate to dissolve;

[0016] Irradiate with UV light for 3 hours to allow for sufficient reaction, and then allow to stand at room temperature to form a freeze-dried powder;

[0017] Accurately weigh 4.5 mg of glucose-6-phosphate dehydrogenase and dissolve it in 0.5 mL of 10 mM PBS solution (pH 7.4). Dissolve 15 mg of the above powder (CsA-BBa) in 0.7 mL of dimethylformamide (DMF) solution. Add CsA-BBa dropwise to the PBS solution containing GDH at 25°C with stirring.

[0018] The pH of the solution was adjusted to 6.0-8.0, and 120 μL of 0.5% EDC was added dropwise, and stirred at 4°C overnight.

[0019] The 6-phosphate glucose dehydrogenase-cyclosporine A conjugate was purified by G-25 gel chromatography and stored at 2-8°C.

[0020] However, existing methods rely on activating the reactive groups of the small molecule drug (cyclosporine A) prior to reacting with the enzyme. This coupling method can result in multiple cyclosporines being linked to the same glucose-6-phosphate dehydrogenase. Furthermore, it is difficult to ensure consistency in the coupling sites, making it difficult to guarantee a targeted 1:1 reaction between the small molecule drug and the enzyme, leading to significant batch-to-batch variability. Summary of the Invention

[0021] In view of the needs in the art, the present application provides a novel 6-phosphate glucose dehydrogenase mutant and its use in preparing a cyclosporine A detection kit.

[0022] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided. Different from the 6-phosphate glucose dehydrogenase mutant disclosed in the previously published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the 6-phosphate glucose dehydrogenase mutant of the present application comprises mutations selected from the group consisting of: D306C, D375C, and G426C.

[0023] According to some embodiments, a 6-phosphate glucose dehydrogenase mutant is provided, wherein the 6-phosphate glucose dehydrogenase mutant is represented by a sequence selected from the group consisting of SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4.

[0024] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0025] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0026] According to some embodiments, a host cell is provided, comprising the expression vector of the present application. The host cell can be a prokaryotic cell (such as a bacterium) or a eukaryotic cell (such as a yeast).

[0027] According to some embodiments, a conjugate is provided, which is formed by coupling the 6-phosphate glucose dehydrogenase mutant of the present application with a hapten in a molar ratio of 1:x. In some embodiments, x is 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some specific embodiments, the 6-phosphate glucose dehydrogenase mutant of the present application and the hapten in a molar ratio of preferably 1:1.

[0028] In some specific embodiments, the molecular weight of the hapten is 100Da to 4000Da, for example: 100, 150, 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 520, 550, 570, 600, 620, 650, 700, 750, 800, 850, 900, 950, 1000, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080, 1090, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1210 000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

[0029] According to the present application, skilled artisans will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as cyclosporin A) that do not themselves carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker to facilitate covalent binding with a sulfhydryl group. Therefore, in the present application, a hapten derivative refers to a hapten that has been modified to carry a sulfhydryl reactive group.

[0030] The hapten is selected from the group consisting of: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0031] Haptens include, but are not limited to, vancomycin, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin and digitoxin), phenolic acid, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, trans-triiodothyronine, free methyl Prostate hormone, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin II, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0032] In specific embodiments, the hapten is cyclosporine A or a derivative thereof.

[0033] In a specific embodiment, the hapten is a cyclosporine A derivative that carries a sulfhydryl-reactive group, such as imide, bromoacetyl, vinyl sulfone, or aziridine.

[0034] In a specific embodiment, the hapten is a cyclosporine A derivative, as shown in Formula I:

[0035]

[0036] Among them, CsA is shown.

[0037] In some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 3, such as 1, 2, or 3.

[0038] In a specific embodiment, the cyclosporine A derivative is represented by Formula III:

[0039]

[0040] Among them, CsA is As shown;.

[0041] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0042] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a cyclosporine A detection reagent.

[0043] According to some embodiments, provided is a use of the conjugate of the present application in preparing a cyclosporine A detection reagent.

[0044] In a specific embodiment, the detection reagent is selected from: enzyme-linked immunosorbent assay detection reagent, chemiluminescence immunoassay detection reagent, homogeneous enzyme immunoassay detection reagent, latex-enhanced immunoturbidimetric detection reagent.

[0045] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0046] According to some embodiments, provided is a use of the conjugate of the present application in preparing a cyclosporine A detection device.

[0047] In a specific embodiment, the detection device can be prepared in the form of a well plate (eg, a 96-well plate), for example, the plate is coated with the reagent according to the present application.

[0048] In a specific embodiment, the detection device can be prepared in the form of particles (such as latex, magnetic beads), for example, the particles are coated with the reagent according to the present application.

[0049] According to some embodiments, a cyclosporine A detection kit is provided, comprising:

[0050] - a first reagent, comprising a substrate, a buffer, and a cyclosporine A antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0051] - a second reagent, comprising the conjugate of the present application and a buffer;

[0052] - optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 ng / ml to 1500 ng / ml cyclosporine A; and

[0053] - Optionally, a quality control comprising 10 mM to 500 mM buffer and 20 ng / ml to 1400 ng / ml cyclosporine A.

[0054] According to one embodiment, a cyclosporine A detection kit is provided, comprising:

[0055] The first reagent comprises:

[0056] 10mM to 500mM buffer,

[0057] 5mM to 50mM substrate,

[0058] 0.1μg / ml to 10μg / ml cyclosporine A antibody,

[0059] 0.05% to 0.5% w / v stabilizer,

[0060] 0.05% to 0.5% w / v surfactant,

[0061] 0.05% to 0.5% w / v preservative;

[0062] A second reagent comprising:

[0063] 10mM to 500mM buffer,

[0064] 0.01 μg / ml to 1 μg / ml of the conjugate according to the present application,

[0065] 0.05% to 0.5% w / v stabilizer,

[0066] 0.05% to 0.5% w / v surfactant,

[0067] 0.05% to 0.5% w / v preservative;

[0068] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, tris(hydroxymethyl)methane buffer; preferably, phosphate buffer; the concentration of the buffer is 10 mmol / L to 500 mmol / L, preferably 50 to 100 mM; the pH of the buffer is 7 to 8.4.

[0069] In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; preferably bovine serum albumin.

[0070] In some embodiments, the surfactant is selected from one or a combination of the following: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween80.

[0071] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, biopreservative PC (such as PC300), thimerosal; the azide is selected from sodium azide and lithium azide.

[0072] In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

[0073] In some specific embodiments, the cyclosporine A antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, or human.

[0074] In some specific embodiments, the cyclosporine A antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment.

[0075] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0076] 1) providing a cyclosporine A derivative according to the present application, in particular providing a cyclosporine A derivative according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0077] 2) providing a 6-phosphate glucose dehydrogenase mutant, preferably providing the 6-phosphate glucose dehydrogenase mutant in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, wherein the buffer has a pH of 6.0 to 8.0);

[0078] 3) contacting the cyclosporine A derivative and the 6-phosphate glucose dehydrogenase mutant at a molar ratio of n:1 at 18° C. to 28° C. for 1 to 4 hours (preferably 2 to 3 hours) to allow the cyclosporine A derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0079] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0080] 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the above values; preferably, n is 20 to 60.

[0081] In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0082] In some specific embodiments, prior to conjugation, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing for a directed reaction with cyclosporine A.

[0083] Wild-type 6-phosphate glucose dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific embodiments, 6-phosphate glucose dehydrogenase is genetically engineered so that the amino acid at a specific site (306, 375 or 426) is mutated to cysteine, thereby carrying a free sulfhydryl group. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 .G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0085] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0086] Figure 3 .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0087] Figure 4 .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0088] Example

[0089] Example 1. Synthesis of cyclosporine A derivatives

[0090]

[0091] 1. Synthesis of Compound 2

[0092] Cyclosporine A (100 mg, 0.08 mmol) was added to a round-bottom flask and dissolved in dry dichloromethane (5 mL). Triethylamine (25 mg, 0.25 mmol) was added to the reaction system and stirred until all the solution was dissolved.

[0093] Oxalyl chloride (52.8 mg, 0.42 mmol) was added to the reaction system, stirred at room temperature, and detected by TLC. The reaction was carried out at room temperature (18-28°C, preferably 20 to 25°C) for about 1 hour. The starting material was basically eliminated. The solvent was removed under reduced pressure, and DCM was used repeatedly (2-3 times) to remove the excess oxalyl chloride, which was used directly in the next step without purification.

[0094] 2. Synthesis of Cyclosporin A Derivatives

[0095] Compound 2 and compound 3 (10 mg, 0.05 mmol) were dissolved in dry dichloromethane (5 mL), and triethylamine (12.4 mg, 0.12 mmol) was added dropwise. The mixture was stirred at room temperature (18-28°C, preferably 20 to 25°C) and monitored by TLC. After completion of the reaction, the product was directly purified using a preparative plate (MeOH / DCM = 1:20) to obtain the cyclosporine A derivative represented by Formula III (45 mg, 38% yield).

[0096] 3. Confirm the product structure by conventional methods. In this example, cyclosporin A has a group that can bind to the enzyme.

[0097] Example 2. Coupling of cyclosporine A derivatives with G6PDH molecules

[0098] 1. Coupling method of the present application

[0099] According to the G6PDH-cyclosporine A conjugate of the present application, the conjugation is carried out in the following manner: the sulfhydryl reactive group (such as but not limited to the maleimide group) on the cyclosporine A derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0100] 1. Dissolve the cyclosporine A derivative prepared in Example 1 in N,N-dimethylformamide (10 mg / ml);

[0101] 2. G6PDH solution: G6PDH (mutant of the present application or mutant of the prior art) dissolved in 0.2 M phosphate buffer, pH 8.0 (2.5 mg / ml enzyme);

[0102] 3. Add 300 μl of glucose 6-phosphate dehydrogenase mutant solution to 50 μl of cyclosporine A derivative solution;

[0103] 4. The mixed solution was shaken thoroughly at room temperature (18-28°C, preferably 20 to 25°C) for 2-3 hours, and then treated with molecular sieve chromatography to obtain the product, namely G6PDH-cyclosporine A conjugate (concentration 0.1 mg / mL-2.5 mg / mL).

[0104] 2. Control Coupling Method

[0105] The G6PDH-cyclosporine A conjugate was prepared according to the method disclosed in the example of CN107782889A.

[0106] Example 3. Preparation of kit

[0107] The following kit for detecting cyclosporine A was prepared, comprising:

[0108] Reagent R1, containing:

[0109] Tris buffer 100 mM, pH 7.4

[0110] 20 mM glucose 6-phosphate

[0111] 20 mM β-nicotinamide adenine dinucleotide

[0112] 1 μg / ml cyclosporine A antibody (commercially available antibody)

[0113] 0.5% w / v bovine serum albumin

[0114] 0.1% w / v Tween80

[0115] 0.1% w / v PC300;

[0116] Reagent R2, including:

[0117] Tris buffer 100 mM, pH 8.2

[0118] 0.05 μg / ml G6PDH-cyclosporine A conjugate

[0119] 0.5% w / v bovine serum albumin

[0120] 0.1% w / v Tween 80

[0121] 0.1% w / v PC300;

[0122] Sample extraction solution: zinc sulfate 50 mM, Tris buffer 50 mM, PC300 0.1% w / v, methanol 50% w / v;

[0123] Calibrators: whole blood matrix, and 0 ng / ml, 50 ng / ml, 300 ng / ml, 600 ng / ml, 900 ng / ml, 1500 ng / ml cyclosporine A (or add as needed);

[0124] Quality control products: whole blood matrix, and 100 ng / ml, 750 ng / ml, 1300 ng / ml cyclosporine A (or add as needed).

[0125] The above reagents (optionally including quality control products and calibrators) are assembled into a detection kit.

[0126] The aforementioned calibrators, quality control materials, and test samples are physiological samples, such as serum, plasma, whole blood, etc. Preferably, the aforementioned calibrators, quality control materials, and test samples are all whole blood matrices and need to be pre-treated with a sample extract before testing.

[0127] Test example

[0128] The kit of the present invention adopts the enzyme amplified immunoassay (EMIT) principle, that is, free CsA in the sample competes with the enzyme-CsA conjugate for binding to the CsA antibody. The more free CsA in the sample, the more antibodies are bound, and the more free conjugate is released. The free conjugate catalyzes the oxidation of β-nicotinamide adenine dinucleotide (NAD + ) is converted into β-nicotinamide adenine dinucleotide (NADH). The CsA concentration in the sample is proportional to the amount of NADH generated. The CsA content can be calculated by the change in the absorbance value at 340nm.

[0129] Table 1. Parameters of fully automatic biochemical analyzer

[0130] model Hitachi 7180 parameters Sample size 2.0 μl Reagent R1 150 μl Reagent R2 50 μl Reading Point 27-34 points Detection wavelength (main) 340nm Detection wavelength (sub) 410nm Curve fitting method Spline Calibrators 0.0, 50.0, 300.0, 600.0, 900.0, 1500.0ng / ml sample Samples to be tested, such as plasma, serum, whole blood, urine, etc.

[0131] Test Example 1. Performance of the kit of this application

[0132] 1. Calibration of absorbance

[0133] Table 2. Calibrated absorbance

[0134]

[0135] 2. Precision Experiment

[0136] Using the calibration curve established above, high, medium and low quality control products and clinical samples were measured.

[0137] Table 3. Total imprecision

[0138]

[0139] 3. Repeatability

[0140] Repeatability experiments were performed by testing the low, medium, and high value quality control samples 20 times each. As shown in the table below, the CVs for the 20 replicates were all within 2.35%.

[0141] Table 4. Reproducibility of the kit

[0142]

[0143] 4. Recycling

[0144] Table 5. Recycling

[0145]

[0146] 5. Linearity of the Detection Kit

[0147] Table 6. Linearity

[0148]

[0149] 6. Accelerated stability of reagents at 37°C

[0150] Table 7. Accelerated stability of reagents at 37°C

[0151]

[0152] 7. Drug Interference Experiment

[0153] The following interfering substances were selected. When the concentration of cyclosporine A was 200-1000 ng / ml, the system deviation produced by the following substances was less than 10%.

[0154] Table 8. Drug interference experiments

[0155] drug Test concentration (μg / mL) Alprazolam 0.57 Digoxin 0.015 Carbamazepine 100 Gentamycin 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 Dafenamide 5.0 Salbutamol 0.15 Metoclopramide 4 nitroglycerin 3.5

[0156] Test Example 2. Antibody Inhibition Rate

[0157] 1. Principle of Antibody Inhibition Rate Detection

[0158] When the antibody binds to the G6PDH-cyclosporine A conjugate, the G6PDH enzyme activity is affected due to steric hindrance, thereby reducing its efficiency in catalyzing the conversion of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental groups with and without the antibody is compared. This difference is reflected in the antibody's ability to inhibit G6PDH.

[0159] 2. Reaction system

[0160] Table 9. Preparation of antibody inhibition rate detection reagents

[0161]

[0162] 3. Results

[0163] By comparing the absorbance values ​​of the G6PDH-cyclosporine A conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0164] Compared to conjugates prepared with previously published mutations (A45C, K55C), the mutants described in this application exhibit significantly improved antibody inhibition rates, reaching over 44% (G426C: 44%; D375C: 50%), with a maximum of 63% (D306C). Previously published mutations (e.g., A45C, K55C) exhibited inhibition rates of 40% to 43%.

[0165] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants (A45C, K55C) in the prior art, the mutation site in the enzyme mutant of the present application (i.e., the site where the free thiol group is introduced) is the location where coupling occurs with the hapten (such as a hormone, a small molecule drug, etc.). When the hapten binds to the hapten-specific antibody at this position, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme. At the same time, after the mutation is introduced, it cannot substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is very important, and it is necessary to take into account the activity of the G6PDH enzyme, the spatial folding of the coupled molecule, and the full exposure of the hapten epitope.

[0166] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the conjugate of the enzyme mutant and cyclosporin A is formulated into a kit, and the reagent has significant performance improvements in terms of inter-batch coefficient of variation, linearity, specificity, etc.

Claims

1. Use of the conjugate in the preparation of a homogeneous enzyme immunoassay reagent for cyclosporine A, wherein: The conjugate is formed by covalently coupling a 6-phosphate glucose dehydrogenase mutant with a cyclosporine A derivative in a molar ratio of 1:1; The cyclosporine A derivative is represented by formula III: in, CsA is As shown in formula II; The 6-phosphate glucose dehydrogenase mutant, compared to the wild-type 6-phosphate glucose dehydrogenase, comprises: a D306C mutation; The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No. 2.

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