A cortisol test kit

Abstract

The present application relates to a cortisol detection kit. Specifically, the 6-phosphogluconate dehydrogenase mutant of the present application comprises one mutation or a combination thereof selected from the following: D306C, D375C, G426C, compared with wild-type 6-phosphogluconate dehydrogenase. The detection kit prepared using the 6-phosphogluconate dehydrogenase mutant of the present application has strong 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 December 31, 2019, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of cortisol detection reagent” (application number 2019114041546). 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 cortisol 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. Once a hapten binds to a protein, it forms an antigenic cluster of that protein. Some substances with smaller molecular weights than typical haptens but with chemically active groups of specific structures (such as penicillin and sulfonamides) are called simple haptens.

[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 cortisol is shown below:

[0007]

[0008] Cortisol, also known as hydrocortisone, or compound F, is an adrenocortical hormone (a type of glucocorticoid) extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism.

[0009] Cortisol is produced from 11-deoxycortisol through the action of 11β-hydroxylase in the mitochondria of the adrenal cortex. Cortisol can also be converted into cortisol by the action of 11-β-hydroxysteroid dehydrogenase.

[0010] Cortisol plays a crucial role in regulating many important physiological processes, including energy metabolism, maintaining electrolyte and blood pressure balance, immune regulation, stress response, cell proliferation and differentiation, memory regulation, and cognitive function. In the blood, cortisol is mainly bound to corticosteroid-binding globulin and albumin, with free cortisol accounting for only 3-5%. Furthermore, cortisol concentration levels exhibit cyclical changes daily, reaching their lowest point in the first half of the night and peaking in the early morning.

[0011] Blood cortisol measurements are primarily used for human diseases such as Cushing's syndrome (excessive cortisol secretion) and Addison's disease (cortisol deficiency), as well as for treatment monitoring (dexamethasone suppression therapy and hormone replacement therapy).

[0012] Currently known methods for cortisol detection mainly include enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay, high-performance liquid chromatography (HPLC), gas-liquid chromatography (GC), gas chromatography (GC), and mass spectrometry (MS). However, these methods all have several drawbacks. For example, although chemiluminescence has good sensitivity, it requires specialized equipment, resulting in high operating costs and hindering its widespread adoption. In clinical diagnostic testing, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetric assay are the primary methods used.

[0013] The principle of homogeneous enzyme immunoassay: In a homogeneous liquid reaction system, enzyme-labeled antigen (such as G6PDH-cortisol) and unlabeled antigen (cortisol) compete for binding with a quantitative amount of antibody (cortisol 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 cortisol in the liquid can be calculated.

[0014] 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.

[0015] The prior art describes a cortisol derivative-G6PDH conjugate and its preparation method:

[0016] 1) Dissolve G6PDH at room temperature in a solution containing Tris, MgCl2 and NaCl (pH = 9.0);

[0017] 2) Add NADH, glucose-6-phosphate, and carbitol;

[0018] 3) Add dimethyl sulfoxide dropwise;

[0019] 4) Weigh out the cortisol derivative under anhydrous conditions and dissolve it in DMF; lower the solution temperature to -2 to -8°C; add tributylamine;

[0020] 5) Add isobutyl chloroformate and stir at -2 to -8°C for 30 minutes;

[0021] 6) Add the activated cortisol derivative solution to the dissolved G6PDH solution dropwise and stir overnight at 2 to 8°C;

[0022] 7) Purify the solution obtained in step 6) by column chromatography to obtain a conjugate of G6PDH-cortisol derivative (e.g., but not limited to the method described in CN105131105A).

[0023] However, existing methods rely on activating the reactive groups of the small molecule drug itself before reacting it with an enzyme. This strategy makes it difficult to guarantee a directional 1:1 reaction between the small molecule drug and the enzyme, resulting in large batch-to-batch variations. Summary of the Invention

[0024] 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 cortisol detection kit.

[0025] 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.

[0026] 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.

[0027] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0028] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0029] 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).

[0030] 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:n.

[0031] In some implementations, n 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.

[0032] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 1:1.

[0033] 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.

[0034] According to this application, those skilled in the art will understand that "hapten" also includes 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., cortisol) 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.

[0035] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0036] Haptens include, but are not limited to: theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin), 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, inverse triiodothyronine, free thyroxine, and so on. The following substances are present in the blood: triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfates, 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, and osteocalcin.

[0037] In a specific implementation plan, the hapten is cortisol or a derivative thereof.

[0038] In a specific implementation, the hapten is a cortisol derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

[0039] In a specific implementation scheme, the hapten is a cortisol derivative, as shown in Formula I:

[0040]

[0041] In some implementations, m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6, such as 1, 2, 3, 4, 5, 6.

[0042] In some implementations, X is maleimide, bromoacetyl, vinyl sulfone, or aziridine.

[0043] Those skilled in the art will understand that the function of X is to react with the thiol group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine, and thiol groups is expected. Although specific groups are used in the examples, they are not intended to be limited thereto.

[0044] In some specific embodiments, the cortisol derivative has a structure selected from the following formula:

[0045]

[0046] m is an integer from 0 to 20, preferably an integer from 1 to 10, and more preferably an integer from 1 to 6.

[0047] In some specific embodiments, the cortisol derivative has a structure selected from the following formula:

[0048]

[0049]

[0050] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0051] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of cortisol detection reagents is provided.

[0052] According to some implementation schemes, the use of the conjugate of this application in the preparation of cortisol detection reagents is provided.

[0053] 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.

[0054] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0055] According to some implementation schemes, the use of the conjugate of this application in the preparation of a cortisol detection device is provided.

[0056] 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.

[0057] 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.

[0058] According to some embodiments, a cortisol detection kit is provided, comprising:

[0059] - First reagent, comprising a substrate, a buffer solution, and a cortisol antibody; said substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0060] - A second reagent, comprising the conjugate and buffer solution of this application;

[0061] -Optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 ng / ml to 800 ng / ml cortisol; and

[0062] -Optionally, a quality control product comprising 10 mM to 500 mM buffer and 100 ng / ml to 500 ng / ml cortisol.

[0063] According to one embodiment, a cortisol detection kit is provided, comprising:

[0064] The first reagent comprises:

[0065] 10mM to 500mM buffer solution

[0066] 5mM to 50mM substrate,

[0067] 10 ng / ml to 10 μg / ml cortisol antibodies,

[0068] 0.1 g / L to 5 g / L stabilizer

[0069] 0.1 g / L to 5 g / L surfactant,

[0070] 0.1 g / L to 5 g / L of preservatives;

[0071] The second reagent comprises:

[0072] 10mM to 500mM buffer solution

[0073] 0.01 μg / ml to 10 μg / ml according to the conjugate of this application,

[0074] 0.1 g / L to 5 g / L stabilizer

[0075] 0.1 g / L to 5 g / L surfactant,

[0076] Preservatives ranging from 0.1 g / L to 5 g / L.

[0077] In some embodiments, the buffer solution is selected from one or a combination of the following: 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 100 mM; the pH of the buffer solution is from 7 to 8.

[0078] 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.

[0079] In some embodiments, the surfactant is selected from one or a combination of the following: Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0080] 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.

[0081] In some embodiments, the substrate comprises: 6-phosphoglucose and β-nicotinamide adenine dinucleotide.

[0082] In some specific implementations, the cortisol antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0083] In some specific implementations, the cortisol antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0084] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0085] 1) Provide cortisol derivatives according to the present application, especially cortisol derivatives according to the present application in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0086] 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);

[0087] 3) At 18°C ​​to 28°C, the glucose-6-phosphate dehydrogenase mutant and the cortisol derivative are contacted at a molar ratio of 1:n for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the cortisol derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate.

[0088] 4) The conjugate may be purified as needed, for example, by desalting.

[0089] In some implementations, the contact molar ratio of enzyme to hapten in the reaction system is 1:n, where n is from 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.

[0090] In other embodiments, the contact molar ratio of enzyme to hapten in the reaction system is 1:n, where n is from 0.01 to 1, for example 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9.

[0091] In some specific implementations, steps 1) and 2) can be interchanged or run in parallel.

[0092] 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 cortisol.

[0093] 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

[0094] Figure 1 Cortisol structural diagram.

[0095] Figure 2 Structural diagram of cortisol derivatives.

[0096] Figure 3A .G6PDH (wild type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0097] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0098] Figure 3C The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0099] Figure 3D The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4).

[0100] Figure 4 Stability when the lid is open. Detailed Implementation

[0101] Example

[0102] Example 1. Synthesis of cortisol derivatives

[0103]

[0104] Cortisol (200 mg, 0.55 mmol) and compound 2 (78 mg, 2.20 mmol) were dissolved in 10 ml of methanol under light-protected conditions and reacted for 5 minutes under light-protected conditions.

[0105] The reaction system was heated to 50°C, and compound 2 (78 mg, 2.20 mmol) and compound 3 (60 mg, 0.55 mmol) were added. The reaction was maintained at this temperature for 5 minutes. The reaction system turned essentially colorless or pale yellow. The solvent was removed under reduced pressure, and the product was purified by column chromatography to obtain 180 mg of product, with a yield of 75%.

[0106] Compound 5 (100 mg, 0.23 mmol) and compound 6 (53 mg, 0.23 mmol) were dissolved in DCM (5 mL), and triethylamine (70 mg, 0.69 mmol) was added dropwise. HATU (105 mg, 0.28 mmol) was then added, and the mixture was stirred at room temperature (18 to 28 °C, preferably 20 to 25 °C) for 5 h. The solvent was removed under reduced pressure to give the cortisol derivative (80 mg, 57%).

[0107] The product structure was confirmed using conventional methods.

[0108] This embodiment gives cortisol a group that can bind to enzymes.

[0109] Example 2. Coupling of cortisol derivatives with G6PDH molecules

[0110] I. Test Methods of This Application

[0111] According to the G6PDH-cortisol conjugate of this application, the coupling is carried out in the following manner: the thiol reactive group (e.g., but not limited to maleimide group) on the cortisol derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0112] 1. Solution preparation:

[0113] Cortisol derivative solution: 10 mg / ml of the cortisol derivative prepared in Example 1 was dissolved in DMF;

[0114] 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;

[0115] Coupling solution: 100mM PB / K, 100mM EDTA, 150mM NaCl, pH=7.2;

[0116] Desalination solution: 100mM PB / K, 0.1% NaN3, 1% NaCl, pH=8.0.

[0117] 2. Coupling operation:

[0118] 2 ml of G6PDH solution, 7.5 ml of coupling solution and 0.5 ml of cortisol derivative solution were reacted at room temperature for 4 h.

[0119] 3. After shaking the above reaction system at room temperature for 4 hours, elute with the above desalting solution using a desalting column, collect the protein peak, and the obtained product is G6PDH-cortisol conjugate.

[0120] II. Control Coupling Method (This experimental procedure is formulated with reference to the method in CN105131105A)

[0121] 1. Weigh 15 mg of G6PDH (100 KU specification) and dissolve it at room temperature in 12 mL of a solution containing 72.6 mg (0.05 M) Tris, 8 mg MgCl2 (3.3 mM) and 100 mg NaCl (pH = 9.0).

[0122] 2. Add 225 mg of reduced nicotinamide adenine dinucleotide (NADH), 135 mg of glucose-6-phosphate, and 0.75 mL of carbitol to the above beaker;

[0123] 3. Add 2 mL of dimethyl sulfoxide dropwise to the above beaker;

[0124] 4. Weigh 10 mg of cortisol derivative under anhydrous conditions and dissolve it in 600 μL of DMF; cool the solution to -2 to -8 °C; add 3 μL of tributylamine;

[0125] 5. Add 1.5 μL of isobutyl chloroformate and stir at -2 to -8°C for 30 minutes;

[0126] 6. Add the activated cortisol derivative solution dropwise to the dissolved G6PDH solution and stir overnight at 2 to 8°C;

[0127] 7. Purify the solution from step 6 using a G-25 gel chromatography column to obtain the final product, which is a glucose-6-phosphate dehydrogenase-hapten conjugate, and store it at 2 to 8°C.

[0128] Example 3. Preparation of the reagent kit

[0129] Prepare the following kit for detecting cortisol, comprising:

[0130] Reagent R1 contains:

[0131] 50mM HEPES, pH 7.0

[0132] 10mM glucose-6-phosphate

[0133] 10mM β-nicotinamide adenine dinucleotide

[0134] 250 ng / ml cortisol antibody (commercially available antibody, no special restrictions)

[0135] 1g / L bovine serum albumin

[0136] 1g / L Tween20

[0137] 1 g / L sodium azide;

[0138] Reagent R2 includes:

[0139] 200mM Tris buffer, pH 8.0

[0140] 0.1 μg / ml G6PDH-Cortisol Conjugate

[0141] 1g / L bovine serum albumin

[0142] 1g / L Tween 20

[0143] 1 g / L sodium azide;

[0144] Calibrator: 20 mM HEPES buffer, and 0.0, 50.0, 100.0, 200.0, 400.0, 800.0 ng / ml cortisol (or add as needed);

[0145] Quality control: 20mM HEPES buffer, and 120ng / ml, 260ng / ml, and 440ng / ml cortisol (or add as needed).

[0146] Assemble the above reagents (optionally including quality control and calibrators) into a homogeneous enzyme immunoassay kit for cortisol.

[0147] Detection example

[0148] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0149] model Hitachi 7180 Analysis points [Rate-A]

[10]

[25]

[34] WAVE(SUB / MAIN)

[410]

[340] S.VIL. [4.0] S.R1

[100] S.R3

[100] ABS.LIMIT:

[32000] [Incrementing] CALIB TYPE: [Spline] POINT: [6]SPAN PONIT[6] Calibrator 0.0, 50.0, 100.0, 200.0, 400.0, 800.0ng / ml sample The samples to be tested are various physiological samples, such as serum and plasma.

[0150] Example 1. Accuracy, precision, and linearity tests of the reagent kit of this application.

[0151] Table 2. Accuracy and Precision (for the D306C mutant)

[0152]

[0153]

[0154] Table 3. Linear

[0155] Test 1 Test 2 Test 3 mean Theoretical value relative deviation absolute deviation 1 5.9 7.6 4.0 5.8 1.53 4.30 2 74.3 73.3 75.5 74.4 72.04 3.2％ 2.32 3 146.5 145.2 142.6 144.8 142.55 1.6％ 2.21 4 217.6 212.7 207.2 212.5 213.06 -0.3％ -0.56 5 283.2 282.0 282.8 282.7 283.57 -0.3％ -0.91 6 344.9 344.1 346.0 345.0 354.08 -2.6％ -9.08 7 408.9 421.3 421.9 417.4 424.59 -1.7％ -7.23 8 489.1 488.3 504.6 494.0 495.11 -0.2％ -1.11 9 562.9 557.7 579.2 566.6 565.62 0.2％ 0.98 10 627.9 655.4 646.6 643.3 636.13 1.1％ 7.17 11 707.0 695.9 723.2 708.7 706.64 0.3％ 2.06 12 782.3 777.9 759.0 773.1 777.15 -0.5％ -4.08 13 835.8 858.3 860.6 851.6 847.66 0.5％ 3.91

[0156] Example 2. Common Drug Interference Prevention

[0157] Table 4. Results of interference resistance assays (for the D306C mutant)

[0158]

[0159]

[0160] Detection Example 3. Correlation

[0161] 1. Test Methods

[0162] One hundred fresh serum samples were collected, and each sample was divided into two aliquots, each with a volume of no less than 500 μl. One aliquot was measured twice using the reagents described in this application (for the 375 mutant) on a Hitachi 7180 instrument, while the other aliquot was measured using a Shimadzu HPLC system. The values ​​obtained by the two methods were analyzed for correlation using scatter plots.

[0163] 2. Experimental Results:

[0164] The resulting function is y = 1.011x + 1.9381, with a correlation coefficient R0. 2 =0.9970.

[0165] The results show that the cortisol concentration in the sample determined by the reagents of this application has a good correlation with the cortisol concentration in the sample determined by HPLC (which can be regarded as the gold standard).

[0166] Table 5. Correlation analysis (unit: ng / ml)

[0167]

[0168]

[0169] Example 4. Inter-batch variation of the cortisol test kit

[0170] Three batches of reagents prepared using the proposed reagent (D306C mutant) and a control reagent were calibrated, and the differences in absorbance variation among the different batches were calculated.

[0171] Table 6. Calibration data between batches

[0172]

[0173] Table 7. Comparison between batches

[0174]

[0175] Example 5. Comparative Analysis of Calibration Stability

[0176] Place the reagent of this application (G426C mutant) and the reagent of the control coupling method separately in the reagent compartment and open the lid ( Figure 4 After calibration, serum samples at three different levels were measured three times at regular intervals for 14 days, and the mean and deviation were calculated.

[0177] Table 8. Calibration stability (unit: ng / ml)

[0178]

[0179] Example 6. Antibody inhibition rate

[0180] 1. Detection principle of antibody inhibition rate

[0181] When the antibody binds to the G6PDH-cortisol 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.

[0182] 2. Reaction system

[0183] Table 9. Preparation of reagents for detecting antibody inhibition rate

[0184]

[0185] 3. Results

[0186] By comparing the absorbance values ​​of the G6PDH-cortisol conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0187] Antibody inhibition rate = (Change in absorbance of G6PDH-cortisol with antibody / Change in absorbance of G6PDH-cortisol without antibody) × 100%.

[0188] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in antibody inhibition rate, reaching over 33% (G426C: 33%; D375C: 48%), with a maximum of 54% (D306C). Previously published mutation sites (e.g., A45C, K55C) showed inhibition rates of 31% and 42%, respectively.

[0189] 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.

[0190] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the formulation of a kit by conjugating the enzyme mutant with cortisol shows a marked improvement in performance in terms of batch-to-batch coefficient of variation, linearity, and specificity.

[0191] Example 7. Alternative Solution

[0192] Referring to the preparation method of Example 3, different test kits and control kits were prepared, the only difference being that the kits prepared in Example 3 were replaced as follows:

[0193] Option 1: Replace the buffer solutions in the first and second reagents with phosphate buffer, glycine buffer, borate buffer, or MOPS buffer in the range of 50 to 100 mM pH 7.0-8.0.

[0194] Option 2: Replace the stabilizers in the first and second reagents with 0.5 to 2.5 g / L of trehalose, sucrose, mannitol, or polyethylene glycol 6000;

[0195] Option 3: Replace the surfactants in the first and second reagents with 0.5 to 2.5 g / L Triton X-100, Tween 80, Brij 35, or Brij 23;

[0196] Option 4: Replace the preservatives in the first and second reagents with lithium azide or PC-300.

[0197] Option 5: Replace compound 2 with compound 3, compound 4, and compound 5 respectively.

[0198] The test kits and control kits from three different batches in the above schemes were tested according to the method in test example 4. The comparison results were similar to those in Tables 6 and 7, showing that the batch-to-batch variation of the test kits was smaller than that of the control kits (data not shown).

Claims

1. A cortisol detection kit, comprising: The first reagent comprises: a substrate, a cortisol antibody, and a buffer solution; The second reagent comprises: a conjugate and a buffer solution; The conjugate is formed by coupling a glucose-6-phosphate dehydrogenase mutant with a cortisol derivative in a molar ratio of 1:

1. The cortisol derivative is shown in Formula II-1: Formula II-1; Compared to wild-type glucose-6-phosphate dehydrogenase, the glucose-6-phosphate dehydrogenase mutant contains the D306C mutation; the glucose-6-phosphate dehydrogenase mutant is shown in SEQ ID No.

2.

2. The cortisol detection kit according to claim 1, further comprising a calibrator containing 10 mM to 500 mM buffer and 0 ng / mL to 800 ng / mL cortisol.

3. The cortisol detection kit according to claim 1, further comprising a quality control, said quality control comprising 10 mM to 500 mM buffer and 100 ng / mL to 500 ng / mL cortisol.

4. The cortisol detection kit according to claim 1, comprising: The first reagent comprises: 10mM to 500mM buffer solution 5mM to 50mM glucose-6-phosphate, 5mM to 50mM oxidized β-nicotinamide adenine dinucleotide, 10 ng / ml to 10 μg / ml cortisol antibody, 0.1 g / L to 5 g / L stabilizer 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L of preservatives; The second reagent comprises: 10mM to 500mM buffer solution The conjugate was prepared at concentrations from 0.01 μg / ml to 10 μg / ml. 0.1 g / L to 5 g / L stabilizer 0.1 g / L to 5 g / L surfactant, 0.1 g / L to 5 g / L of preservatives; The buffer solutions in the first reagent and the second reagent are each independently selected from one or a combination of the following: phosphate buffer, glycine buffer, Tris buffer, borate buffer, MOPS buffer, HEPES buffer; The pH of the buffer solution in the first reagent and the second reagent is independently between 7 and 8; The stabilizers in the first reagent and the second reagent are each independently 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; The surfactants in the first reagent and the second reagent are each independently 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, and AEO7; The preservatives in the first reagent and the second reagent are each independently selected from one or a combination of the following: azide, MIT, biological preservative PC, and thimerosal.

5. The cortisol detection kit according to claim 4, comprising: The first reagent comprises: 100 mM to 300 mM buffer solution 10 mM to 20 mM glucose-6-phosphate, 10 mM to 20 mM oxidized β-nicotinamide adenine dinucleotide, 100 ng / ml to 500 ng / ml cortisol antibodies, 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives; The second reagent comprises: 100 mM to 300 mM buffer solution The conjugate was prepared at concentrations of 0.05 μg / ml to 0.5 μg / ml. 1g / L to 5g / L stabilizer 1g / L to 5g / L surfactant, 1 g / L to 5 g / L of preservatives.

6. The cortisol detection kit according to claim 4, wherein: The stabilizer is bovine serum albumin.

7. The cortisol detection kit according to claim 4, wherein: The surfactant is Tween20.

8. The cortisol detection kit according to claim 4, wherein: The preservative is selected from one or a combination of the following: sodium azide, lithium azide, and PC-300.

9. The cortisol detection kit according to claim 4, comprising: The first reagent comprises: 50 mM HEPES, pH 7.0 10mM glucose-6-phosphate, 10mM oxidized β-nicotinamide adenine dinucleotide, 250 ng / ml cortisol antibody, 1 g / L bovine serum albumin 1 g / L Tween20 1 g / L sodium azide; The second reagent comprises: 200mM Tris buffer, pH 8.0 0.1 μg / ml of the conjugate, 1 g / L bovine serum albumin 1 g / L Tween20 1 g / L sodium azide.

Images