Methods of preparing conjugates

Abstract

The present application relates to a preparation method of a conjugate. Specifically, the 6-phosphogluconate dehydrogenase mutant of the present application comprises one mutation or a combination thereof selected from D306C, D375C, G426C compared with wild-type 6-phosphogluconate dehydrogenase. The detection kit prepared by using the 6-phosphogluconate dehydrogenase mutant of the present application has high specificity, high sensitivity, convenient operation, short detection time, accurate quantification, and is suitable for high-throughput detection.

Description

[0001] This application is a divisional application of application number 2020100131747, filed on January 7, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of cyclosporine A detection reagent”. Technical Field

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

[0003] Haptens are certain small molecules (molecular weight less than 4000 Da) that, when alone, cannot induce an immune response (i.e., they lack immunogenicity). However, when they cross-link or bind with large protein molecules or non-antigenic carriers such as polylysine, they acquire immunogenicity and induce an immune response. These small molecules can bind to response effect products and thus possess antigenicity. They only exhibit immunoreactivity but lack immunogenicity; they are also known as incomplete antigens.

[0004] A hapten is an antigen that can bind to a corresponding antibody to produce an antigen-antibody reaction, but cannot independently stimulate the production of antibodies in humans or animals. It only has immunoreactivity, not immunogenicity, and is also called an 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 stimulate the animal to produce corresponding antibodies.

[0005] Small molecule antigens or haptens lack two or more sites suitable for sandwich assays, therefore they cannot be detected using the double-antibody sandwich method; instead, a competitive mode is often used. The principle is that the antigen in the sample and a certain amount of enzyme-labeled antigen compete for binding to the solid-phase antibody. The higher the antigen content in the sample, the less enzyme-labeled antigen binds to the solid phase, resulting in a lighter color development. This method is commonly used for the ELISA assay of small molecule hormones and drugs.

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

[0007]

[0008] Cyclosporine A (CsA) is a lipid-soluble cyclic peptide composed of various amino acids secreted by fungi (Trichoderma polysporum or Trichoderma columnarum). As an immunosuppressant with high selectivity and ultra-low myelotoxicity, CsA selectively inhibits the proliferation and release of T helper lymphocytes. Simultaneously, this drug possesses antifungal activity, effectively reducing infection rates in patients. Cyclosporine A has significant clinical application value in organ and tissue transplantation, hematological diseases, and ophthalmic diseases. In organ and tissue transplantation, it can prevent rejection reactions in allogeneic kidney, liver, heart, and bone marrow transplants, and can also prevent and treat graft-versus-host disease during bone marrow transplantation. In hematological diseases, it can be used for aplastic anemia, erythrocytic aplastic anemia, myelodysplastic syndrome, and autoimmune hemolytic anemia. In ophthalmic diseases, it is mainly used for Behçet's syndrome, dry eye syndrome, scleritis, and allergic conjunctivitis.

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

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

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

[0012] Currently known methods for detecting cyclosporine A mainly include high-performance liquid chromatography (HPLC), luminescent immunoassay, and enzyme-linked immunosorbent assay (ELISA). HPLC requires complex sample pretreatment, is complicated and time-consuming, and is expensive; luminescent immunoassay reagents are expensive, unsuitable for routine therapeutic drug detection, and even less conducive to widespread adoption.

[0013] Existing homogeneous enzyme immunoassay and latex agglutination turbidimetric assays are often limited in application due to their complex preparation processes and large batch-to-batch variations.

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

[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 dissolve by sonication;

[0016] After 3 hours of ultraviolet light irradiation, the reaction was complete, and the mixture was allowed to stand at room temperature to form a lyophilized 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). Take 15 mg of the above powder (CsA-BBa) and dissolve it in 0.7 mL of dimethylformamide (DMF) solution. Under constant temperature of 25 °C and stirring, add CsA-BBa dropwise to the PBS solution containing GDH.

[0018] Adjust the pH of the solution to 6.0-8.0, then add 120 μL of 0.5% carbodiimide (EDC) dropwise, and stir overnight at 4°C.

[0019] The glucose-6-phosphate 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) itself before reacting it with the enzyme. This coupling method can result in multiple cyclosporine A molecules linked to the same glucose-6-phosphate dehydrogenase, and the coupling sites are difficult to ensure consistency, making it difficult to guarantee a directional 1:1 reaction between the small molecule drug and the enzyme, leading to large batch-to-batch variations. Summary of the Invention

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

[0022] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. Unlike the published glucose-6-phosphate dehydrogenase mutant in US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose-6-phosphate dehydrogenase mutant of this application contains mutations selected from the following: D306C, D375C, and G426C.

[0023] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided, said glucose-6-phosphate dehydrogenase mutant being selected from the sequences shown below: SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4.

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

[0025] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

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

[0027] According to some embodiments, a conjugate is provided, which is formed by conjugating the glucose-6-phosphate dehydrogenase mutant of this application with a hapten at 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, 10. In some specific embodiments, the molar ratio of the glucose-6-phosphate dehydrogenase mutant of this application to the hapten is preferably 1:1.

[0028] In some specific implementations, the molecular weight of the hapten is from 100 Da to 4000 Da, 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, 1 000, 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 this application, those skilled in the art will understand that "hapten" also includes its derivative forms. To facilitate coupling with glucose-6-phosphate dehydrogenase, haptens that do not inherently possess a coupling group (e.g., a group that reacts with a thiol group) (e.g., cyclosporine A) can be modified to have a linker for covalently binding with a thiol group. Therefore, in this application, a hapten derivative refers to a hapten modified to possess a thiol-reactive group.

[0030] Haptens are selected from: small molecule drugs (such as antibiotics and 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 acids, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic acid metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma glycoproline, 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, transtriiodothyronine, free thyroxine. Hormones, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, adrenaline, 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 a specific implementation plan, the hapten is cyclosporine A or a derivative thereof.

[0033] In a specific implementation, the hapten is a cyclosporine A derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

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

[0035]

[0036] Among them, CsA is As shown.

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

[0038] In a specific implementation scheme, the cyclosporine A derivative is shown in Formula III:

[0039]

[0040] Among them, CsA is As shown;

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

[0042] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of cyclosporine A detection reagent is provided.

[0043] According to some implementation schemes, the use of the conjugate of this application in the preparation of cyclosporine A detection reagent is provided.

[0044] In the specific implementation plan, the detection reagents are selected from: enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay (CIA) reagents, homogeneous enzyme immunoassay (HIA) reagents, and latex-enhanced immunoturbidimetric assay (LTIA) reagents.

[0045] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0046] According to some implementation schemes, the use of the conjugate of this application in the preparation of a cyclosporine A detection device is provided.

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

[0048] In a specific implementation, the detection device can be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with the reagent according to this application.

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

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

[0051] - A second reagent, comprising the conjugate and buffer solution of this application;

[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 sample 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 solution

[0057] 5mM to 50mM substrate,

[0058] 0.1 μg / ml to 10 μg / 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] The second reagent comprises:

[0063] 10mM to 500mM buffer solution

[0064] 0.01 μg / ml to 1 μg / ml according to the conjugate of this 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 solution is selected from one or a combination of the following: TAPS, glycerol buffer, phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolpropane buffer; preferably, phosphate buffer; the concentration of the buffer solution is from 10 mmol / L to 500 mmol / L, preferably from 50 to 100 mM; the pH of the buffer solution is from 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, and 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: azides, MIT, biological preservatives PC (such as PC300), and thimerosal; the azides are selected from sodium azide and lithium azide.

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

[0073] In some specific implementations, the cyclosporine A antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0074] In some specific implementations, the cyclosporine A antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0075] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0076] 1) Provide cyclosporine A derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

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

[0078] 3) At 18°C ​​to 28°C, the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate;

[0079] 4) The conjugate may be purified as needed, for example, by desalting.

[0080] In some embodiments, the contact molar ratio of enzyme to hapten in the reaction system is 1:n, where n is from 1 to 500, for example, 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 any of the above values; preferably n is from 20 to 60.

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

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

[0083] Wild-type glucose-6-phosphate dehydrogenase does not contain a free sulfhydryl group. Therefore, in some specific implementations, glucose-6-phosphate dehydrogenase is genetically engineered to mutate the amino acid at a specific site (positions 306, 375, or 426) to cysteine, thereby giving it a free sulfhydryl group. Attached Figure Description

[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 The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0087] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0088] Example

[0089] Example 1. Synthesis of Cyclosporine A Derivative

[0090]

[0091] 1. Synthesis of Compound 2

[0092] Add cyclosporine A (100 mg, 0.08 mmol) to a round-bottom flask and dissolve it in dry dichloromethane (5 mL). Add triethylamine (25 mg, 0.25 mmol) to the reaction system and stir until completely dissolved.

[0093] Add oxalyl chloride (52.8 mg, 0.42 mmol) to the reaction system, stir at room temperature, detect by TLC, and react at room temperature (18-28℃, preferably 20 to 25℃) for about 1 hour until the starting material is basically gone. Remove the solvent under reduced pressure, and use DCM repeatedly (2-3 times) to remove excess oxalyl chloride, which can be used directly in the next step without purification.

[0094] 2. Synthesis of Cyclosporine A Derivatives

[0095] Compounds 2 and 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 20-25 °C) and detected by TLC. After the reaction was complete, the mixture was directly purified by preparative plate (MeOH / DCM = 1:20) to give the cyclosporine A derivative of Formula III (45 mg, yield 38%).

[0096] 3. The product structure was confirmed using conventional methods. In this example, cyclosporine A contains a group that can bind to the enzyme.

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

[0098] I. Coupling Method of This Application

[0099] According to the G6PDH-cyclosporine A conjugate of this application, the conjugation is carried out in the following manner: the thiol reactive group (such as, but not limited to, maleimide group) on the cyclosporine A derivative molecule is covalently bonded to the thiol 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 (the mutant of this application or the prior art mutant) is dissolved in 0.2M 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 above mixed solution is shaken thoroughly at room temperature (18-28℃, preferably 20-25℃) for 2-3 hours, and then subjected to molecular sieve chromatography. The resulting product is G6PDH-cyclosporine A conjugate (concentration 0.1mg / mL-2.5mg / mL).

[0104] II. Contrastive Coupling Method

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

[0106] Example 3. Preparation of the reagent kit

[0107] Prepare the following kit for the detection of cyclosporine A, comprising:

[0108] Reagent R1 contains:

[0109] Tris buffer 100mM, pH 7.4

[0110] 20mM glucose-6-phosphate

[0111] 20mM 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 includes:

[0117] Tris buffer 100mM, 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 50mM, Tris buffer 50mM, PC300 0.1% w / v, methanol 50% w / v;

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

[0124] Quality control materials: whole blood matrix, and cyclosporine A at concentrations of 100 ng / ml, 750 ng / ml, and 1300 ng / ml (or added as needed).

[0125] Assemble the above reagents (optionally including quality control samples and calibrators) into a test kit.

[0126] The calibrators, quality control samples, and test samples mentioned above are physiological samples, such as serum, plasma, and whole blood. Preferably, the calibrators, quality control samples, and test samples mentioned above are all whole blood matrix, and pretreatment with sample extraction solution is required before testing.

[0127] Detection example

[0128] This invention's kit utilizes the Enzyme Amplified Immunoassay (EMIT) principle, where free CsA in the sample competitively binds to CsA antibodies against an enzyme-CsA conjugate. The more free CsA in the sample, the more antibodies bind, and the more free conjugate is released. The released conjugate catalyzes the oxidation of β-nicotinamide adenine dinucleotide (NAD). + CsA is converted into β-nicotinamide adenine dinucleotide (NADH). The concentration of CsA in the sample is directly proportional to the amount of NADH generated. The content of CsA can be calculated by the change in absorbance at 340 nm.

[0129] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0130]

[0131]

[0132] Example 1. Performance of the reagent kit of this application

[0133] 1. Calibration absorbance

[0134] Table 2. Calibration Absorbance

[0135]

[0136] 2. Precision Experiment

[0137] Using the calibration curves established above, high, medium, and low quality control products and clinical samples were measured.

[0138] Table 3. Total Imprecision

[0139]

[0140] 3. Repeatability

[0141] Repeatability tests were conducted on low, medium, and high-value quality control samples, 20 times each. As shown in the table below, the CV values ​​were all within 2.35% after 20 repeated tests.

[0142] Table 4. Reproducibility of the kit

[0143]

[0144] 4. Recycling

[0145] Table 5. Recycling

[0146]

[0147]

[0148] 5. Linearity of the detection kit

[0149] Table 6. Linear

[0150]

[0151] 6.37℃ reagent accelerated stability

[0152] Table 7. Accelerated stability of reagents at 37℃

[0153]

[0154]

[0155] 7. Drug Interference Experiment

[0156] The following interfering substances were selected. When the concentration of cyclosporine A was 200-1000 ng / ml, the systematic deviation produced by the following substances was less than 10%.

[0157] Table 8. Drug Interference Experiment

[0158] drug Test concentration (μg / mL) Alprazolam 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 Aminopteridine 5.0 Salbutamol 0.15 Metoclopramide 4 nitroglycerin 3.5

[0159] Example 2. Antibody inhibition rate

[0160] 1. Detection principle of antibody inhibition rate

[0161] When the antibody binds to the G6PDH-cyclosporine A conjugate, the steric hindrance affects the activity of the G6PDH enzyme, 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 can be compared. This difference reflects the inhibitory ability of the antibody on G6PDH.

[0162] 2. Reaction system

[0163] Table 9. Preparation of reagents for detecting antibody inhibition rate

[0164]

[0165] 3. Results

[0166] By comparing the absorbance values ​​of the G6PDH-cyclosporine A conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0167] Compared to conjugates prepared using previously published mutation sites (A45C, K55C), the mutants in this application show a significant improvement in antibody inhibition rate, reaching over 44% (G426C: 44%; D375C: 50%), with a maximum of 63% (D306C). Previously published mutation sites (e.g., A45C, K55C) have inhibition rates of 40% to 43%.

[0168] While not limited to specific theories, this can be partially explained as follows: Compared to existing G6PDH mutants (A45C, K55C), the mutation site (i.e., the site introducing a free thiol group) in the enzyme mutant of this application is the location where it couples with haptens (such as hormones, small molecule drugs, etc.). When a hapten binds to a hapten-specific antibody at this site, the resulting steric hindrance has the greatest impact on the activity of the G6PDH enzyme, while the introduction of the mutation does not substantially affect the spatial folding of the molecule. Therefore, the location of this mutation site is crucial, requiring consideration of G6PDH enzyme activity, the spatial folding of the coupled molecule, and the sufficient exposure of the hapten epitope.

[0169] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the reagent kit formulated by conjugating the enzyme mutant with cyclosporine A shows a significant improvement in performance in terms of batch-to-batch coefficient of variation, linearity, and specificity.

Claims

1. A method for preparing a coupling agent, comprising the steps of: 1) Provides cyclosporine A derivatives; 2) Provide a glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant is coupled with the cyclosporine A derivative; The cyclosporine A derivative is shown in Formula III: in, CsA is As shown; The glucose-6-phosphate dehydrogenase mutant, compared to the wild-type glucose-6-phosphate dehydrogenase, contains the D306C mutation; The glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No.

2.

2. The method for preparing the coupling compound according to claim 1, comprising the steps of: 1) The cyclosporine A derivative is provided in an aprotic solvent; 2) Provide the glucose-6-phosphate dehydrogenase mutant in a buffer solution; 3) The glucose-6-phosphate dehydrogenase mutant and the cyclosporine A derivative are contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate. 4) Purify the conjugate; Steps 1) and 2) can be interchanged or performed in parallel; The buffer solution is selected from: PBS, Tris, TapS, and TAPSO. The pH of the buffer solution is 6.0 to 8.0; The aprotic solvent is selected from: acetonitrile, dimethylformamide, and dimethyl sulfoxide; Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306.

3. In the method for preparing the coupling compound according to claim 2, in step 3): The glucose-6-phosphate dehydrogenase mutant and the cyclosporine A derivative were contacted at 20°C to 25°C for 2 to 3 hours to couple the cyclosporine A derivative and the glucose-6-phosphate dehydrogenase mutant to obtain the conjugate.

4. In the method for preparing the coupling compound according to claim 2, in step 4): The conjugate was then desalted and purified.

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