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 Chinese patent application filed on January 6, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of tacrolimus detection reagent” (application number 2020100095702). 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 tacrolimus 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] Tacrolimus, a specific example of a hapten, has the following structural formula:

[0007]

[0008] Tacrolimus, also known as FK506, is a macrolide antibiotic. It was discovered in Japan in 1984 and first used clinically as an immunosuppressant in 1989. As an immunosuppressant, tacrolimus is highly lipophilic, incompletely absorbed, and unstable.

[0009] Tacrolimus has a narrow therapeutic range of safe and effective treatments. Insufficient dosage or low blood concentrations of tacrolimus may lead to transplant rejection. Excessive tacrolimus concentrations can cause serious adverse reactions, including nephrotoxicity, neurotoxicity, post-transplant diabetes, increased susceptibility to infection, carcinogenesis, hypertension, and gastrointestinal disturbances.

[0010] For the reasons mentioned above, monitoring tacrolimus blood concentration is an effective way to assist clinical treatment, improve treatment efficacy, and reduce toxicity risks.

[0011] Currently known methods for tacrolimus detection 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.

[0012] 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.

[0013] Prior art CN108107200A describes a tacrolimus detection kit, which discloses a method for preparing glucose-6-phosphate dehydrogenase and tacrolimus conjugate:

[0014] Dissolve 20-100 mg of tacrolimus in methanol, add anhydrous sodium acetate, dissolve evenly, add carboxymethyl hydroxylamine, dissolve and mix well, heat under nitrogen protection and react overnight, then distill under reduced pressure to obtain a waxy substance, add dimethylformamide to dissolve, filter to remove precipitate, and distill under reduced pressure to remove solvent to obtain product A;

[0015] Dissolve 10-50 mg of product A in 20-100 mL of dimethylformamide, then slowly add 50-150 μL of carbodiimide (EDC) dropwise to the above solution while stirring, and mix by vortexing for 60-150 minutes;

[0016] Dissolve 10-50 mg of glucose-6-phosphate dehydrogenase (100-300 KU) in PBS buffer and shake well.

[0017] - Slowly add the tacrolimus solution to solution 3 with stirring, and stir for 8-16 hours to obtain glucose-6-phosphate dehydrogenase and tacrolimus conjugate.

[0018] However, existing methods rely on activating the reactive groups of the small molecule drug (tacrolimus) itself before reacting with the enzyme. This coupling method can result in multiple tacrolimus 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

[0019] 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 tacrolimus detection kit.

[0020] 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, G426C, and D375C.

[0021] 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.

[0022] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0023] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0024] 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).

[0025] 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.

[0026] In some implementations, x is 1 to 50, 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.

[0027] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 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., tacrolimus) can be modified to have a linker for covalent 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 the specific implementation plan, the hapten is tacrolimus or its derivative.

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

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

[0035]

[0036] In some implementations, m is an integer from 1 to 10, preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

[0037] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0038] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of tacrolimus detection reagents is provided.

[0039] According to some implementation schemes, the use of the conjugates of this application in the preparation of tacrolimus detection reagents is provided.

[0040] 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.

[0041] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0042] According to some implementation schemes, the use of the conjugate of this application in the preparation of a tacrolimus detection device is provided.

[0043] 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.

[0044] 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.

[0045] According to some implementation schemes, a tacrolimus detection kit is provided, comprising:

[0046] - First reagent, comprising a substrate, a buffer solution, and a tacrolimus antibody; the substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0047] - A second reagent, comprising the conjugate and buffer solution of this application;

[0048] -Optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 ng / ml to 30 ng / ml tacrolimus; and

[0049] -Optionally, a quality control sample comprising 10 mM to 500 mM buffer solution and 5 ng / ml to 25 ng / ml tacrolimus.

[0050] According to one embodiment, a tacrolimus detection kit is provided, comprising:

[0051] The first reagent comprises:

[0052] 10mM to 500mM buffer solution

[0053] 5mM to 50mM substrate,

[0054] 0.1 μg / ml to 10 μg / ml mg / L tacrolimus antibody,

[0055] 0.1 g / L to 5 g / L stabilizer

[0056] 0.1 g / L to 5 g / L surfactant,

[0057] 0.1 g / L to 5 g / L of preservatives;

[0058] The second reagent comprises:

[0059] 10mM to 500mM buffer solution

[0060] 0.1 μg / ml to 10 μg / ml according to the conjugate of this application,

[0061] 0.1 g / L to 5 g / L stabilizer

[0062] 0.1 g / L to 5 g / L surfactant,

[0063] 0.1 g / L to 5 g / L of preservatives;

[0064] The third reagent consists of a mixture of methanol and ethanol in a volume ratio of 3:1, and 0.5-5% zinc sulfate.

[0065] In some embodiments, the buffer 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 is from 10 mmol / L to 500 mmol / L, preferably from 50 to 100 mM; the pH of the buffer is from 7 to 8.

[0066] 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.

[0067] 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 Tween20.

[0068] In some embodiments, the preservative is selected from one or a combination of the following: azides, MIT, biological preservatives PC (such as PC-300), and thimerosal; the azides are selected from: sodium azide and lithium azide.

[0069] In some embodiments, the substrate comprises: 6-phosphoglucose and β-nicotinamide adenine dinucleotide.

[0070] In some specific implementation schemes, the tacrolimus antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0071] In some specific implementation schemes, the tacrolimus antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0072] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0073] 1) Provide tacrolimus derivatives according to this application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0074] 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);

[0075] 3) At 18°C ​​to 28°C, the tacrolimus 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 tacrolimus derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

[0076] 4) The conjugate may be purified as needed, for example, by desalting.

[0077] 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 50.

[0078] In some specific implementations, steps 1) and 2) can be interchanged or run in parallel.

[0079] In some specific implementations, prior to coupling, the glucose-6-phosphate dehydrogenase contains one or more free sulfhydryl groups, thereby allowing a directed reaction with tacrolimus.

[0080] 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

[0081] Figure 1 .G6PDH (wild-type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0082] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0083] Figure 3 The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0084] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0085] Example

[0086] Example 1. Synthesis of tacrolimus derivatives

[0087]

[0088] Where m = 1.

[0089] Tacrolimus (100 mg, 0.11 mmol) was added to a round-bottom flask and dissolved in dry DCM (5 mL). A catalytic equivalent of 4-N,N-dimethylpyridine was added, along with DCC (27 mg, 0.13 mmol). The mixture was stirred under nitrogen protection until completely dissolved. 4-maleimidebutyric acid (20 mg, 0.11 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature (18°C to 28°C, preferably 20°C to 25°C) for approximately 4 hours. The reaction was monitored by TLC. After completion, the mixture was directly purified using a preparative plate (MeOH / DCM = 1:20) to finally obtain the tacrolimus derivative (51 mg, 47% yield).

[0090] The product structure was confirmed using conventional methods.

[0091] This embodiment enables tacrolimus to have a group that can bind to enzymes.

[0092] Example 2. Coupling of tacrolimus derivatives with G6PDH molecules

[0093] I. Coupling Method of This Application

[0094] According to the G6PDH-tacrolimus conjugate of this application, the coupling is carried out in the following manner: the thiol reactive group (such as, but not limited to, maleimide group) on the tacrolimus derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0095] 1. The tacrolimus derivative prepared in Example 1 was dissolved in N,N-dimethylformamide (10 mg / ml);

[0096] 2. G6PDH solution: G6PDH (the mutant of this application or the prior art mutant) is dissolved in 100 mmol PB, 100 mmol NaCl, pH=8.0, 5 mg / mL enzyme;

[0097] 3. Add 2 ml of glucose-6-phosphate dehydrogenase mutant solution, 7.5 ml of PB solution, and 0.5 ml of tacrolimus derivative solution;

[0098] 4. The above mixed solution is shaken thoroughly at room temperature (18-28℃, preferably 20 to 25℃) for 2-3 hours, and then desalted (desalting solution: 100mM PB, 0.1% NaN3, 1% NaCl, pH=8.0). The protein peak is collected, and the product obtained is the G6PDH-tacrolimus conjugate.

[0099] II. Contrastive Coupling Method

[0100] The G6PDH-tacrolimus conjugate was prepared according to the method disclosed in the examples of CN108107200A:

[0101] 1. Dissolve 20-100 mg of tacrolimus in methanol, add anhydrous sodium acetate, dissolve evenly, add carboxymethyl hydroxylamine, dissolve and mix well, heat under nitrogen protection and react overnight, then distill under reduced pressure to obtain a waxy substance, add dimethylformamide to dissolve, filter to remove precipitate, and distill under reduced pressure to remove solvent to obtain product A;

[0102] 2. Dissolve 10-50 mg of product A in 20-100 mL of dimethylformamide, and then slowly add 50-150 μL of carbodiimide (EDC) dropwise to the above solution while stirring, and mix by vortexing for 60-150 minutes;

[0103] 3. Dissolve 10-50 mg of glucose dehydrogenase (100-300 KU) in PBS buffer and shake well.

[0104] 4. Slowly add the tacrolimus solution from step 2 to the solution from step 3 while stirring, and stir for 8-16 hours.

[0105] Example 3. Preparation of the reagent kit

[0106] Prepare the following kit for detecting tacrolimus, comprising:

[0107] Reagent R1 contains:

[0108] HEPES buffer 50mM, pH 7.0

[0109] 10mM glucose-6-phosphate

[0110] 10mM β-nicotinamide adenine dinucleotide

[0111] 1 μg / ml tacrolimus antibody (commercially available antibody)

[0112] 1g / L bovine serum albumin

[0113] 1g / L Tween20

[0114] 1 g / L sodium azide;

[0115] Reagent R2 includes:

[0116] 200mM Tris buffer, pH 8.0

[0117] 1 μg / ml G6PDH-tacrolimus conjugate

[0118] 1g / L bovine serum albumin

[0119] 1g / L Tween 20

[0120] 1 g / L sodium azide;

[0121] Sample extraction solution: a mixture of methanol and ethanol at a volume ratio of 3:1, and 1% zinc sulfate;

[0122] Calibrator: 20 mM HEPES buffer, and 0.0, 2.5, 5.0, 10.0, 20.0, 30.0 ng / ml tacrolimus (or add as needed);

[0123] Quality control: 20mM HEPES buffer, and 8.1ng / ml, 15.4ng / ml, 23.2ng / ml (or add as needed).

[0124] Assemble the above reagents (optionally including quality control and calibrators) into a homogeneous tacrolimus enzyme immunoassay kit.

[0125] Detection example

[0126] The principle of homogeneous enzyme immunoassay: In a homogeneous liquid reaction system, enzyme-labeled antigen (such as G6PDH-tacrolimus) and unlabeled antigen (tacrolimus) compete for binding with a quantitative amount of antibody (tacrolimus antibody). The more the antibody binds to the unlabeled antigen, the more activity of the enzyme-labeled antigen is released, and the more NADH is generated from the substrate NAD+ by the enzyme. By detecting the change in absorbance of NADH at a wavelength of 340 nm, the content of tacrolimus in the liquid can be calculated.

[0127] Mix the whole blood sample with the quality control and calibrators thoroughly. Use a pipette to transfer 200 μl of the sample into the corresponding centrifuge tube. Add an equal volume of sample extraction solution and immediately cap the tube. Vortex the tube for at least 10 seconds to ensure thorough mixing. Centrifuge at 12,000 rpm for 5 minutes. Transfer each supernatant to a small tube and cap it tightly. The sample is ready for testing.

[0128] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0129] model Hitachi 7180 Specifications Analysis points [Rate-A]

[10]

[25]

[34] WAVE(SUB / MAIN)

[410]

[340] S.VIL.

[20] S.R1

[150] S.R3

[50] ABS.LIMIT:

[32000] [Incrementing] CALIB TYPE: [Spline] POINT: [6]SPAN PONIT[6] Calibrator 0.0, 2.5, 5.0, 10.0, 20.0, 30.0ng / ml sample Samples to be tested, such as plasma, serum, whole blood, urine, etc.

[0130] Example 1. Performance of the reagent kit of this application

[0131] 1. Calibration absorbance

[0132] Table 2. Calibration Absorbance

[0133]

[0134] 2. Precision Experiment

[0135] Using the calibration curves established above, high, medium, and low quality control products and clinical samples were measured.

[0136] Table 3. Total Imprecision

[0137]

[0138] 3. Repeatability

[0139] Table 4. Box repeatability

[0140]

[0141] 4. Recycling

[0142] Table 5. Recycling

[0143]

[0144]

[0145] 5. Tacrolimus Assay Kit Linearity

[0146] Table 6. Linear

[0147]

[0148]

[0149] 6. Tacrolimus 37℃ reagent accelerated stability

[0150] After acceleration at 37°C for 7 days, the calibrated absorbance of the reagent in this application decreased by approximately 16%, while that of the control reagent decreased by approximately 51% after acceleration at 37°C for 7 days.

[0151] Table 7. Accelerated stability of reagents at 37℃

[0152]

[0153] Example 2. Antibody inhibition rate

[0154] 1. Detection principle of antibody inhibition rate

[0155] When the antibody binds to the G6PDH-tacrolimus 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.

[0156] 2. Reaction system

[0157] Table 8. Preparation of reagents for detecting antibody inhibition rate

[0158]

[0159] 3. Results

[0160] By comparing the absorbance values ​​of the G6PDH-tacrolimus conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0161] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in antibody inhibition rate, reaching over 45% (G426C: 45%; D375C: 57%), with a maximum of 58% (D306C). Previously published mutation sites (e.g., A45C, K55C) had inhibition rates of 41% and 25%, respectively.

[0162] Table 9. Antibody inhibition rates of different G6PDH mutants

[0163]

[0164] 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.

[0165] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the formulation of a kit using the enzyme mutant conjugate with tacrolimus showed a marked 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) Provide tacrolimus derivatives; 2) Provide a glucose-6-phosphate dehydrogenase mutant; 3) The glucose-6-phosphate dehydrogenase mutant is coupled to the tacrolimus derivative at a molar ratio of 1:1; The tacrolimus derivative is shown in Formula I: in, m is an integer from 1 to 6; The glucose-6-phosphate dehydrogenase mutant, compared to the wild-type glucose-6-phosphate dehydrogenase, contains a D306C or D375C mutation, and the glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No. 2 or SEQ ID No.

3.

2. The method for preparing the coupling compound according to claim 1, wherein: The value of m is 1.

3. The method for preparing the coupling compound according to claim 1, comprising the steps of: 1) The tacrolimus 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 tacrolimus derivative are contacted at 18°C ​​to 28°C for 1 hour to 4 hours to couple the tacrolimus 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 one or a combination of the following: acetonitrile, dimethylformamide, and dimethyl sulfoxide; Prior to step 3), the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306 or 375.

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