Tacrolimus Test Kit

Abstract

This application relates to a tacrolimus detection kit. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to the wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. The detection kit prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibits high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making it suitable for high-throughput detection.

Description

[0001] This application is a divisional application of Chinese Patent Application “6-phosphogluconate dehydrogenase mutant and its use in preparing tacrolimus detection reagent” (application number 2020100095702) filed on January 6, 2020. TECHNICAL FIELD

[0002] The present application relates to the field of biological detection, in particular to a mutant enzyme 6-phosphogluconate dehydrogenase (abbreviated as G6PDH) and its application in a tacrolimus detection kit. BACKGROUND

[0003] Hapten, certain small molecule substances (molecular weight less than 4000 Da), which alone cannot induce immune response, i.e. not immunogenic, but when it is cross-linked or combined with macromolecular protein or non-antigenic polylysine carrier, it can obtain immunogenicity and induce immune response. These small molecule substances can be combined with response effect products and have antigenicity, which only has immunoreactivity and not immunogenicity, also known as incomplete antigen.

[0004] Hapten can bind to corresponding antibodies to produce antigen-antibody reaction, and cannot alone stimulate human or animal body to produce antibodies. It only has immunoreactivity and not immunogenicity, also known as incomplete antigen. Most polysaccharides, lipids, hormones, and small molecule drugs belong to haptens. If the haptens are combined with a certain protein molecule (carrier) by chemical methods, new immunogenicity can be obtained, and the corresponding antibodies can be stimulated in animals.

[0005] Small molecule antigens or haptens lack two or more than two sites that can be used for sandwich method, so they cannot be determined by double antibody sandwich method, and are mostly used in competition mode. The principle is that the antigens in the sample and a certain amount of enzyme-labeled antigens compete for binding to the solid-phase antibody. The more the amount of antigens in the sample, the less the enzyme-labeled antigens bound to the solid phase, and the lighter the color. Small molecule hormones, drugs, etc. ELISA determination mostly uses this method.

[0006] Tacrolimus (tacrolimus) as a specific example of hapten, the structural formula is as follows:

[0007]

[0008] Tacrolimus, also known as FK506, is a macrolide antibiotic. Tacrolimus was discovered in Japan in 1984, and was first used in clinical practice as an immunosuppressant in 1989. Tacrolimus, as an immunosuppressant, has high lipophilicity, incomplete absorption and instability.

[0009] The safe and effective therapeutic range of tacrolimus is narrow, and insufficient dosage of tacrolimus or too low blood concentration of tacrolimus can lead to rejection of transplantation. Too high concentration of tacrolimus can lead to serious adverse reactions, including nephrotoxicity, neurotoxicity, post-transplant diabetes, increased susceptibility to infection, cancer, hypertension, and gastrointestinal dysfunction.

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

[0011] The known methods for detecting tacrolimus mainly include high performance liquid chromatography (HPLC), luminescence immunoassay, and enzyme-linked immunosorbent assay (ELISA). The HPLC method requires complex sample pretreatment, complicated operation, long cycle, and high cost; the luminescence immunoassay method has high reagent cost and is not suitable for routine drug detection, let alone wide application.

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

[0013] The prior art CN108107200A describes a tacrolimus detection kit, in which a preparation method of 6-phosphogluconate dehydrogenase and tacrolimus conjugate is disclosed:

[0014] - 20-100 mg of tacrolimus is dissolved in methanol, anhydrous sodium acetate is added, and after uniform dissolution, carboxymethyl hydroxylamine is added, mixed uniformly, and then warmed under nitrogen protection overnight, then distilled under reduced pressure to obtain a waxy substance, dissolved in dimethylformamide, filtered to remove the precipitate, and distilled under reduced pressure to remove the solvent to obtain product A;

[0015] - 10-50 mg of product A is dissolved in 20-100 mL of dimethylformamide, and 50-150 μL of carbodiimide (EDC) is slowly added to the above solution under stirring, and rotated for 60-150 minutes;

[0016] - 10-50 mg of 6-phosphogluconate dehydrogenase with a specification of 100-300 KU is dissolved in PBS buffer and shaken uniformly;

[0017] - The tacrolimus solution is slowly added to the 3 solution under stirring, and stirred for 8-16 hours to obtain the 6-phosphogluconate dehydrogenase and tacrolimus conjugate.

[0018] However, the prior art method relies on the activation of the reactive group on the small molecule drug (tacrolimus) itself, and then reacts with the enzyme. Such coupling method may have multiple tacrolimus linked to the same glucose-6-phosphate dehydrogenase, and the coupling site is difficult to ensure consistency, and it is difficult to ensure the directional 1:1 reaction between the small molecule drug and the enzyme, resulting in large batch-to-batch differences. SUMMARY

[0019] In view of the needs in the art, the present application provides a novel 6-phosphogluconate dehydrogenase mutant and its use in preparing a tacrolimus detection kit.

[0020] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided. The 6-phosphogluconate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of D306C, G426C, and D375C.

[0021] According to some embodiments, a 6-phosphogluconate dehydrogenase mutant is provided, which is shown in 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 encoding the 6-phosphogluconate dehydrogenase mutant of the present application is provided.

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

[0024] According to some embodiments, a host cell comprising the expression vector of the present application is provided. 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 a 6-phosphogluconate dehydrogenase mutant of the present application coupled with a hapten at a molar ratio of 1: x.

[0026] In some embodiments, 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 embodiments, the 6-phosphogluconate dehydrogenase mutant of the application is coupled to the hapten in a molar ratio of preferably 1:1.

[0028] In some specific embodiments, the hapten has a molecular weight of 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, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000.

[0029] According to the application, the skilled person will understand that "hapten" also comprises forms of derivatives thereof. In order to facilitate the coupling to the 6-phosphogluconate dehydrogenase, for those haptens that do not carry a coupling group (e.g. a group that reacts with a thiol group) themselves (e.g. tacrolimus), the hapten can be engineered to carry a linker in order to covalently bind to a thiol group. Thus, in the present application, a hapten derivative refers to a hapten that has been engineered to carry a thiol-reactive group.

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

[0031] Haptens such as, but not limited to, vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1,25 dihydroxy vitamin D, folic acid, cardiac glycosides (including digoxin, digitoxin), enzyme phenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the urinary nicotinic metabolite cotinine, urinary morphine, urinary monohydroxyphenyl derivatives, neuropeptide tyrosine, plasma cholinesterase, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocortical hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, reverse triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17a hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-keto prostaglandin F1a, prostacyclin, epinephrine, catecholamines, norepinephrine, cholecystokinin, natriuretic, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive intestinal peptide, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0032] In a particular embodiment, the hapten is tacrolimus or a derivative thereof.

[0033] In a particular embodiment, the hapten is a tacrolimus derivative which bears a thiol-reactive group such as a maleimide, a bromoacetyl group, a vinyl sulfone or an aziridine.

[0034] In a particular 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, there is provided a reagent comprising the conjugate of the application.

[0038] According to some embodiments, there is provided the use of a 6-phosphogluconate dehydrogenase mutant of the application for the manufacture of a tacrolimus detection reagent.

[0039] According to some embodiments, there is provided the use of a conjugate of the application for the manufacture of a tacrolimus detection reagent.

[0040] In particular embodiments, the detection reagent is selected from the group consisting of: an enzyme-linked immunoassay reagent, a chemiluminescent immunoassay reagent, a homogeneous enzyme immunoassay reagent, a latex-enhanced immunoturbidimetric reagent.

[0041] In particular embodiments, the detection reagent is preferably a reagent based on a competition assay.

[0042] According to some embodiments, there is provided the use of the conjugate of the application for the preparation of a tacrolimus detection device.

[0043] In particular embodiments, the detection device can be prepared in the form of a well plate (e.g. a 96-well plate), such as a plate coated with the reagent according to the application on the plate.

[0044] In particular embodiments, the detection device can be prepared in the form of a particle (e.g. latex, magnetic beads), such as a particle coated with the reagent according to the application on the particle.

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

[0046] - a first reagent comprising a substrate, a buffer and a tacrolimus antibody; the substrate being a substrate for 6-phosphogluconate dehydrogenase;

[0047] - a second reagent comprising the conjugate of the 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 quality control comprising 10 mM to 500 mM buffer, 5 ng / ml to 25 ng / ml tacrolimus.

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

[0051] a first reagent comprising:

[0052] 10 mM to 500 mM buffer,

[0053] 5 mM to 50 mM substrate,

[0054] 0.1 pg / ml to 10 pg / 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 preservative;

[0058] a second reagent comprising:

[0059] 10 mM to 500 mM buffer,

[0060] 0.1 pg / ml to 10 pg / ml conjugate according to the present 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 preservative;

[0064] a third reagent comprising: 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: TAPS, BES buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbitone 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: 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: Brij 23, Brij 35, Triton X-100, Triton X-405, Tween 20, Tween 30, Tween 80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.

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

[0069] In some embodiments, the substrate comprises: 6-phosphogluconate, beta-nicotinamide adenine dinucleotide.

[0070] In some specific embodiments, the tacrolimus antibody is derived from: mouse, rat, cat, dog, primate, bovine, equine, ovine, camelid, avian, human.

[0071] In some specific embodiments, the tacrolimus antibody is selected from: mono-antibody, poly-antibody, recombinant antibody, chimeric antibody, antigen-binding fragment.

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

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

[0074] 2) providing a 6-phosphogluconate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment, such as but not limited to PBS, Tris, TAPS, TAPSO, with a pH of 6.0 to 8.0);

[0075] 3) contacting the tacrolimus derivative and the 6-phosphogluconate dehydrogenase mutant at a molar ratio of n: 1 at 18°C to 28°C for 1 hour to 4 hours (preferably 2 hours to 3 hours) so that the tacrolimus derivative and the 6-phosphogluconate dehydrogenase mutant are coupled to obtain the seed conjugate;

[0076] 4) optionally purifying the seed conjugate, such as desalting treatment, etc. as needed.

[0077] In some embodiments, the molar ratio of enzyme and hapten in the reaction system is 1: n, wherein n is 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 range between any of the above-mentioned 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, the 6-phosphogluconate dehydrogenase contains one or more free sulfhydryl groups before coupling, allowing directed reaction with tacrolimus.

[0080] Wild-type 6-phosphogluconate dehydrogenase does not contain free sulfhydryl group, thus in some specific embodiments, the 6-phosphogluconate dehydrogenase is genetically engineered so that the amino acid at a specific site (position 306, 375 or 426) is mutated to cysteine, thus bearing a free sulfhydryl group. BRIEF DESCRIPTION OF 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 derivative

[0087]

[0088] wherein m = 1.

[0089] Into a round bottom flask, tacrolimus (100 mg, 0.11 mmol) was dissolved in dry DCM (5 mL), a catalytic equivalent of 4-N,N-dimethylpyridine was added, and DCC (27 mg, 0.13 mmol) was added, and stirred under nitrogen protection until completely dissolved. To the reaction system, 4-maleimide butyric acid (20 mg, 0.11 mmol) was added, and stirred at room temperature (18-28°C, preferably 20-25°C) for about 4 h, and detected by TLC. The reaction was directly purified by preparative plate (MeOH / DCM = 1:20), and finally tacrolimus derivative (51 mg, yield 47%) was obtained.

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

[0091] This example makes tacrolimus bear a group that can bind to enzyme.

[0092] Example 2. Coupling of tacrolimus derivative with G6PDH molecule

[0093] I. Coupling method of the present application

[0094] The G6PDH-tacrolimus conjugate according to the present application is coupled in the following way: the thiol-reactive group (such as but not limited to a maleimide group) on the tacrolimus derivative molecule covalently binds 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 prior art mutant) is dissolved in PB 100 mmol, NaCl 100 mmol, pH = 8.0, 5 mg / mL enzyme;

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

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

[0099] II. Control coupling method

[0100] Prepare the G6PDH-tacrolimus conjugate according to the method disclosed in the example of CN108107200A:

[0101] 1. Dissolve 20-100 mg of tacrolimus in methanol, add anhydrous sodium acetate, dissolve uniformly, then add carboxymethyl hydroxylamine, dissolve and mix uniformly, warm under nitrogen protection overnight, then distill under reduced pressure to obtain a waxy substance, dissolve in dimethylformamide, filter to remove the precipitate, and distill the solvent under reduced pressure 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 carbodiimide (EDC) to the above solution under stirring, and rotate mix for 60-150 minutes;

[0103] 3. Take 10-50 mg of glucose dehydrogenase with a specification of 100-300 KU, and dissolve in PBS buffer and shake uniformly;

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

[0105] Example 3. Preparation of a kit

[0106] A kit for detecting tacrolimus is prepared, which comprises:

[0107] Reagent R1, comprising:

[0108] HEPES buffer 50 mM, pH 7.0

[0109] 10 mM 6-phosphogluconate

[0110] 10 mM β-nicotinamide adenine dinucleotide

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

[0112] 1 g / L bovine serum albumin

[0113] 1 g / L Tween 20

[0114] 1 g / L sodium azide;

[0115] Reagent R2, comprising:

[0116] 200 mM Tris buffer, pH 8.0

[0117] 1 μg / ml G6PDH-tacrolimus conjugate

[0118] 1 g / L bovine serum albumin

[0119] 1 g / L Tween 20

[0120] 1 g / L sodium azide;

[0121] Sample extraction solution: methanol and ethanol mixed 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 added as needed);

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

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

[0125] Detection example

[0126] Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-tacrolimus) competes with non-labeled antigen (tacrolimus) for binding with quantitative antibody (tacrolimus antibody). The more the antibody binds with the non-labeled antigen, the more the enzyme-labeled antigen is released, and the more NADH is generated by the enzyme catalyzing the substrate NAD+. The absorbance change of NADH at 340 nm wavelength is detected, and the tacrolimus content in the liquid can be calculated.

[0127] Mix the human whole blood sample and the quality control and calibration completely, take 200 μl of the sample into the corresponding centrifuge tube with a pipette, immediately cover the cap after adding an equal volume of sample extraction solution, shake on a vortex shaker for at least 10 seconds to ensure that the sample is fully mixed, centrifuge at a speed of 12000 r / min for 5 minutes on a centrifuge, transfer each supernatant to a small tube and tightly cap the cap, and the sample can be used for detection.

[0128] Table 1. Parameters of automatic biochemical analyzer

[0129] Model Hitachi 7180 parameters Analysis point [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] Calibrator 0.0, 2.5, 5.0, 10.0, 20.0, 30.0 ng / ml Sample Sample to be tested, such as plasma, serum, whole blood, urine, etc.

[0130] Detection example 1. Performance of the kit of the application

[0131] 1. Calibration absorbance

[0132] Table 2. Calibration absorbance

[0133]

[0134] 2. Precision experiment

[0135] Using the calibration curve established as above, high, medium and low quality control and clinical samples are determined.

[0136] Table 3. Total imprecision

[0137]

[0138] 3. Reproducibility

[0139] Table 4. Box reproducibility

[0140]

[0141] 4. Recovery

[0142] Table 5. Recovery

[0143]

[0144]

[0145] 5. Tacrolimus detection kit linearity

[0146] Table 6. Linear

[0147]

[0148]

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

[0150] The calibration absorbance of the reagent of the present application decreased by about 16% after 7 days of 37°C acceleration, while the calibration absorbance of the control reagent decreased by about 51% after 7 days of 37°C acceleration.

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

[0152]

[0153] Detection Example 2. Antibody inhibition rate

[0154] 1. Detection principle of antibody inhibition rate

[0155] When the antibody binds to the G6PDH-tacrolimus conjugate, the G6PDH enzyme activity is affected due to steric hindrance, thereby reducing the efficiency of its catalysis of NAD to NADH. By detecting the change in the amount of NADH, the difference between the experimental group with and without the addition of the antibody can be compared, which reflects the inhibitory ability of the antibody on G6PDH.

[0156] 2. Reaction system

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

[0158]

[0159] 3. Results

[0160] By comparing the absorbance measurements of G6PDH-tacrolimus conjugate with and without the addition of the antibody, the inhibition of the antibody on G6PDH can be obtained.

[0161] Compared with the published mutation site (A45C), the mutants of the present application have a significant improvement in antibody inhibition rate, which can reach more than 45% (G426C: 45%; D375C: 57%), and the highest is 58% (D306C). The inhibition rates of previously published mutation sites (such as A45C, K55C) are 41% and 25%.

[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 (i.e., the site where the free thiol group is introduced) in the enzyme mutant of the present application is the position where the hapten (such as hormones, small molecule drugs, etc.) is coupled. When the hapten is bound to the hapten-specific antibody at this position, the steric hindrance formed has the greatest effect on the activity of G6PDH enzyme, and at the same time, after introducing the mutation, it cannot substantially affect the spatial folding of the molecule. Therefore, the position of this mutation site is very important, and it needs to take into account the activity of G6PDH enzyme, the spatial folding of the coupled molecule, and the sufficient exposure of the hapten epitope.

[0165] Due to the significant improvement in antibody inhibition rate of the enzyme mutant, the conjugate prepared by coupling the enzyme mutant with tacrolimus is formulated into a kit, and the reagent has obvious performance improvement in batch variation coefficient, linearity, specificity and other performance.

Claims

1. A tacrolimus detection kit comprising: a first reagent comprising: a substrate, a tacrolimus antibody, and a buffer; a second reagent comprising: a conjugate and a buffer; wherein, the conjugate is a 6-phosphogluconate dehydrogenase mutant covalently coupled with a tacrolimus derivative at a molar ratio of 1:1; the tacrolimus derivative is shown in Formula I: wherein, m is 1; the 6-phosphogluconate dehydrogenase mutant comprises a D306C or D375C mutation compared to a wild-type 6-phosphogluconate dehydrogenase; and the 6-phosphogluconate dehydrogenase mutant is shown in SEQ ID No. 2 or SEQ ID No.

3. 2.The tacrolimus detection kit according to claim 1, wherein: a first reagent comprising: 10mM to 500mM buffer, 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 0.1μg / ml to 10μg / ml anti-tacrolimus antibody, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, and 0.1g / L to 5g / L preservative; a second reagent comprising: 10mM to 500mM buffer, 0.1μg / ml to 10μg / ml the conjugate, 0.1g / L to 5g / L stabilizer, 0.1g / L to 5g / L surfactant, and 0.1g / L to 5g / L preservative; the kit further comprises a third reagent comprising: a mixture of methanol and ethanol at a volume ratio of 5:1 to 2:1, and 0.5% to 5% zinc sulfate by mass / volume; the buffer is selected from any one or a combination of TAPS buffer, phosphoric acid buffer, glycine buffer, Tris buffer, boric acid buffer, MOPS buffer, and HEPES buffer; the pH of the buffer is 7 to 8; the stabilizer is selected from any one or a combination of bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; the surfactant is selected from any one or a combination of Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, and AEO7; the preservative is selected from any one or a combination of azide, MIT, biological preservative PC, and thiomersal. 3.The tacrolimus detection kit according to claim 2, wherein: a first reagent comprising: HEPES buffer 50mM, PH 7.0, 10mM glucose-6-phosphate, 10mM oxidized β-nicotinamide adenine dinucleotide, 1μg / ml tacrolimus antibody, 1g / L bovine serum albumin, 1g / L Tween20, and 1g / L sodium azide; a second reagent comprising: 200mM Tris buffer, pH 8.0, 1 μg / ml of the conjugate, 1 g / L bovine serum albumin, 1 g / L Tween 20, and 1 g / L sodium azide; a third reagent comprising: a mixture of methanol and ethanol at a volume ratio of 3:1, and 0.5% to 5% zinc sulfate by mass / volume.

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