Use of the conjugate in the preparation of a detection reagent

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 Chinese patent application "6-Phosphate Glucose Dehydrogenase Mutant and Its Use in the Preparation of Tacrolimus Detection Reagent" (Application No. 2020100095702) filed on January 6, 2020. Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a mutant enzyme glucose-6-phosphate dehydrogenase (G6PDH for short) and its application in a tacrolimus 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] Tacrolimus is a specific example of a hapten, and its structural formula is shown below:

[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, with incomplete and unstable absorption.

[0009] Tacrolimus has a narrow therapeutic range for safety and effectiveness. Inadequate tacrolimus dosing or low blood concentrations can lead to transplant rejection. Excessive tacrolimus concentrations can cause serious adverse reactions, including nephrotoxicity, neurotoxicity, post-transplant diabetes, increased susceptibility to infection, cancer, hypertension, and gastrointestinal disorders.

[0010] Based on the above reasons, monitoring of tacrolimus blood concentration is an effective way to assist clinical treatment, improve treatment effects, and reduce toxicity risks.

[0011] Currently known tacrolimus detection methods 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 therapeutic drug testing and hindering widespread adoption.

[0012] The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to their complex preparation processes and large batch differences.

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

[0014] Dissolve 20-100 mg of tacrolimus in methanol, add anhydrous sodium acetate, and after uniform dissolution, add carboxymethylhydroxylamine, dissolve and mix thoroughly. Heat and react overnight under nitrogen protection, then distill under reduced pressure to obtain a waxy product. Add dimethylformamide to dissolve, filter to remove the precipitate, and distill under reduced pressure to remove the 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 EDC to the solution under stirring, and rotate and mix for 60-150 minutes;

[0016] - Dissolve 10-50 mg of 100-300 KU 6-phosphate glucose dehydrogenase in PBS buffer and shake evenly;

[0017] - The tacrolimus solution was slowly added to solution 3 under stirring, and the mixture was stirred for reaction for 8-16 hours to obtain a conjugate of glucose-6-phosphate dehydrogenase and tacrolimus.

[0018] However, existing methods rely on activating reactive groups within the small-molecule drug (tacrolimus) prior to reaction with the enzyme. This coupling method can result in multiple tacrolimus molecules 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

[0019] 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 tacrolimus detection kit.

[0020] 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, G426C, and D375C.

[0021] 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.

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

[0023] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0024] 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).

[0025] 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 at a molar ratio of 1:x.

[0026] 47, 48, 49, 50. In some embodiments, x is 1 to 50, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50.

[0027] In some specific embodiments, the molar ratio of the 6-phosphate glucose dehydrogenase mutant of the present application to the hapten is 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 derivatives thereof. To facilitate coupling with glucose-6-phosphate dehydrogenase, haptens (such as tacrolimus) that do not inherently carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker for covalent binding to the 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 tacrolimus or a derivative thereof.

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

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

[0035]

[0036] In some embodiments, 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, which comprises the conjugate of the present application.

[0038] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a tacrolimus detection reagent.

[0039] According to some embodiments, provided is a use of the conjugate of the present application in preparing a tacrolimus detection reagent.

[0040] 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.

[0041] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0042] According to some embodiments, provided is a use of the conjugate of the present application in preparing a tacrolimus detection device.

[0043] 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.

[0044] 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.

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

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

[0047] - a second reagent, comprising the conjugate of the present application and a buffer;

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

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

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

[0051] A first reagent comprising:

[0052] 10mM to 500mM buffer,

[0053] 5mM to 50mM substrate,

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

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

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

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

[0058] A second reagent comprising:

[0059] 10mM to 500mM buffer,

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

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

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

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

[0064] The third reagent comprises: 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, 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.

[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, 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: azide, MIT, biopreservative PC (such as PC-300), thimerosal; the azide is selected from sodium azide and lithium azide.

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

[0070] In some specific embodiments, the tacrolimus antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, or human.

[0071] In some specific embodiments, the tacrolimus antibody is selected from the group consisting of: monoclonal antibody, polyclonal antibody, recombinant antibody, chimeric antibody, and antigen-binding fragment.

[0072] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0073] 1) providing a tacrolimus derivative according to the present application, in particular providing a tacrolimus derivative according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0074] 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);

[0075] 3) contacting the tacrolimus 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 tacrolimus derivative and the 6-phosphate glucose dehydrogenase mutant to couple to obtain the conjugate;

[0076] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0077] 47, 48, 49, 50, 100, 200, 300, 400, 500, and ranges between any of the above values; preferably, n is 20 to 50.

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

[0079] 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 tacrolimus.

[0080] 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

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

[0084] Figure 4 .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[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, followed by DCC (27 mg, 0.13 mmol). Under nitrogen, the mixture was stirred until completely dissolved. 4-Maleimidobutyric acid (20 mg, 0.11 mmol) was added to the reaction system and stirred at room temperature (18°C to 28°C, preferably 20°C to 25°C) for approximately 4 h. The mixture was then monitored by TLC. After completion of the reaction, the product was directly purified using a preparative plate (MeOH / DCM = 1:20) to obtain a tacrolimus derivative (51 mg, 47% yield).

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

[0091] This embodiment provides tacrolimus with a group that can bind to an enzyme.

[0092] Example 2. Coupling of Tacrolimus Derivatives with G6PDH Molecules

[0093] 1. Coupling method of the present application

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

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

[0096] 2. G6PDH solution: G6PDH (mutant of the present application or mutant of the prior art) was 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 mixed solution was thoroughly shaken at room temperature (18-28°C, preferably 20 to 25°C) for 2-3 hours, desalted (desalting solution: 100 mM PB, 0.1% NaN3, 1% NaCl, pH = 8.0), and the protein peak was collected. The resulting product was G6PDH-tacrolimus conjugate.

[0099] 2. Control Coupling Method

[0100] G6PDH-tacrolimus conjugate was prepared by referring to the method disclosed in Example 108107200A:

[0101] 1. Dissolve 20-100 mg of tacrolimus in methanol, add anhydrous sodium acetate, and after uniform dissolution, add carboxymethylhydroxylamine, dissolve and mix thoroughly. Heat and react overnight under nitrogen protection, then distill under reduced pressure to obtain a waxy product. Add dimethylformamide to dissolve, filter to remove the precipitate, and distill under reduced pressure to remove the solvent to obtain Product A.

[0102] 2. Dissolve 10-50 mg of product A in 20-100 mL of dimethylformamide, then slowly add 50-150 μL of EDC to the solution under stirring, and rotate to mix for 60-150 minutes;

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

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

[0105] Example 3. Preparation of kit

[0106] The following tacrolimus detection kit was prepared, comprising:

[0107] Reagent R1, containing:

[0108] HEPES buffer 50 mM, pH 7.0

[0109] 10 mM glucose 6-phosphate

[0110] 10 mM β-nicotinamide adenine dinucleotide

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

[0112] 1g / L bovine serum albumin

[0113] 1g / L Tween20

[0114] 1g / L sodium azide;

[0115] Reagent R2, including:

[0116] 200 mM Tris buffer, pH 8.0

[0117] 1 μg / ml G6PDH-tacrolimus conjugate

[0118] 1g / L bovine serum albumin

[0119] 1g / L Tween 20

[0120] 1g / L sodium azide;

[0121] Sample extract: a mixture of methanol and ethanol in 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 added as needed);

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

[0124] The above reagents (optionally including quality control products and calibrators) are assembled into a tacrolimus homogeneous enzyme immunoassay kit.

[0125] Test example

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

[0127] Thoroughly mix the human whole blood sample, quality control product, and calibrator. Use a pipette to take 200 μl of the sample into the corresponding centrifuge tube. Immediately cover the tube with the lid after adding an equal volume of sample extract. Vortex the tube on a vortex shaker for at least 10 seconds to ensure that the sample is fully mixed. Centrifuge at 12,000 rpm for 5 minutes. Transfer each supernatant to a small tube and seal the lid tightly. The sample can be used for testing.

[0128] Table 1. Parameters of fully automatic biochemical analyzer

[0129] model Hitachi 7180 parameters Analysis Points [Rate-A]

[10]

[25]

[34] WAVE(SUB / MAIN)

[410]

[340] S.VIL.

[20] S.R1

[150] S.R3

[50] ABS.LIMIT:

[32000] [Incremental] CALIB TYPE: [Spline] POINT: [6]SPAN PONIT[6] Calibrators 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] Test Example 1. Performance of the kit of this application

[0131] 1. Calibration of absorbance

[0132] Table 2. Calibrated absorbance

[0133]

[0134] 2. Precision experiment

[0135] Using the calibration curve 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 Detection Kit Linearity

[0146] Table 6. Linearity

[0147]

[0148]

[0149] 6. Tacrolimus 37℃ reagent accelerated stability

[0150] After the reagent of the present application is accelerated at 37°C for 7 days, the absorbance of the calibration decreases by about 16%, and after the control reagent is accelerated at 37°C for 7 days, the absorbance of the calibration decreases by about 51%.

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

[0152]

[0153] Test Example 2. Antibody Inhibition Rate

[0154] 1. Principle of Antibody Inhibition Rate Detection

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

[0156] 2. Reaction system

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

[0158]

[0159] 3. Results

[0160] By comparing the absorbance values of the G6PDH-tacrolimus conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0161] Compared to the previously published mutation site (A45C), the mutants of this application have significantly improved antibody inhibition rates, reaching over 45% (G426C: 45%; D375C: 57%), with a maximum of 58% (D306C). Previously published mutation sites (such as A45C and K55C) had inhibition rates of 41% and 25% respectively.

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

[0163]

[0164] 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.

[0165] Because the enzyme mutant has a significant improvement in antibody inhibition rate, the reagent kit, after the enzyme mutant is coupled with tacrolimus, has a significant improvement in the inter-batch coefficient of variation, linearity, specificity and other performance.

Claims

1. Use of the conjugate in the preparation of a detection reagent, wherein: The detection reagent is a detection reagent for tacrolimus; The detection reagent is a homogeneous enzyme immunoassay detection reagent; The conjugate is formed by covalently coupling a 6-phosphate glucose dehydrogenase mutant with a tacrolimus derivative at a molar ratio of 1:1; The tacrolimus derivative is represented by formula I: Formula I, in, m is 1; The 6-phosphate glucose dehydrogenase mutant comprises any one of the following mutations compared to the wild-type 6-phosphate glucose dehydrogenase: D306C, D375C; and The 6-phosphate glucose dehydrogenase mutant is shown in SEQ ID No. 2 or SEQ ID No. 3.

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