6-Phosphate Glucose Dehydrogenase Mutant and Its Use in Preparing Detection Reagents

Abstract

This application relates to mutants of 6-glucose phosphate dehydrogenase and their use in preparing detection reagents. Specifically, the 6-glucose phosphate dehydrogenase mutants of the present application contain 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 mutants of the present application has strong specificity, high sensitivity, easy operation, short detection time, and accurate quantitative determination, making it suitable for high-throughput detection.

Description

[0001] This application claims priority to patent application No. 201910017764.4 filed on January 9, 2019 and patent application No. 201910423122.4 filed on May 21, 2019. This application is a divisional application of patent application No. 2019113721472 filed on December 27, 2019, entitled "6-Phosphate Glucose Dehydrogenase Mutants and Their Use in the Preparation of Detection Reagents." Technical Field

[0002] The present application relates to the field of biological detection, and in particular to a multi-site mutated enzyme 6-phosphate glucose dehydrogenase (G6PDH) and its application in a 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 the production of antibodies in humans or animals on its own. It is only immunoreactive and 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 certain protein molecule (carrier), it will acquire new immunogenicity and can stimulate animals to produce corresponding antibodies. Once a hapten binds to a protein, it forms an antigenic cluster of that protein. Some substances with smaller molecular weight than ordinary haptens but with specific chemically active groups (such as penicillin and sulfonamides) are called simple haptens.

[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] Cholyglycin (CG), as a specific example of a hapten, is a conjugated bile acid formed by the combination of bile acid and glycine. It is one of the main components of bile acid. Cholesterol undergoes a series of complex enzyme-catalyzed reactions in liver cells to form primary bile acids, including cholic acid (CA) and chenodeoxycholic acid (CDCA). The steroid nucleus of bile acid has three hydroxyl groups (C3, C7, C12). The hydroxyl group at the end of the side chain is bound to glycine via a peptide bond to form cholic acid ( Figure 1 ).

[0007] Glycocholic acid is synthesized by liver cells and discharged into the gallbladder via bile ductules and bile ducts. It then enters the duodenum along with bile, aiding in the digestion and absorption of dietary fat. 95% of bile acid is reabsorbed in the ileum and colon, returning to the liver via the portal vein. It is then taken up and reused by liver cells, and the reabsorbed glycocholic acid reenters the enterohepatic circulation. Through this mechanism, the body can fully utilize glycocholic acid.

[0008] Under normal circumstances, the amount of bile acid in peripheral blood is extremely low. Whether fasting or postprandial, the level of glycocholic acid in the blood of healthy individuals remains very low. When liver cells are damaged or bile accumulates, glycocholic acid metabolism and circulation become disrupted, reducing the liver cells' ability to absorb glycocholic acid and leading to elevated blood levels of glycocholic acid. The level of glycocholic acid is correlated with the severity of liver cell damage and bile acid metabolism disorders.

[0009] Measuring serum glycocholic acid is a sensitive indicator for evaluating liver cell function and hepatobiliary circulation. Compared with conventional liver function tests such as ALT, AST, total bilirubin (TBIL), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), and serum albumin (ALB), glycocholic acid measurement is more sensitive. Therefore, glycocholic acid can be used as a more effective indicator in liver function tests for conditions such as chronic hepatitis, acute hepatitis, cirrhosis, liver cancer, obstructive liver disease, enterohepatic circulation disorders, and bile duct and gallbladder excretion disorders.

[0010] Currently known methods for detecting glycocholic acid mainly include radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLI), high performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), gas chromatography-mass spectrometry (GC-MS), etc. However, these methods all have many drawbacks. For example, the radioimmunoassay isotope has many drawbacks such as radioactive contamination, a short shelf life, and inconvenient operation. The ELISA is relatively cumbersome and time-consuming to operate, making it unsuitable for clinical use. Although chemiluminescence has better sensitivity, it requires supporting special equipment, and the high cost of use is not conducive to its promotion. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex-enhanced immunoturbidimetry are the main detection methods.

[0011] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, the enzyme-labeled antigen (such as G6PDH-CG) competes with the unlabeled antigen (CG) for binding with a quantitative antibody (CG antibody). The more the antibody binds to the unlabeled antigen, the more activity the enzyme-labeled antigen releases, and the more NADH the enzyme catalyzes from the substrate NAD+ to generate NADH. By detecting the absorbance change of NADH at a wavelength of 340nm, the content of CG in the liquid can be inferred. Summary of the Invention

[0012] 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 glycocholic acid detection kit.

[0013] 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.

[0014] 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.

[0015] According to some embodiments, a polynucleotide is provided, which encodes the 6-phosphate glucose dehydrogenase mutant of the present application.

[0016] According to some embodiments, an expression vector is provided, comprising the polynucleotide of the present application.

[0017] 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).

[0018] 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 in a molar ratio of 1:n.

[0019] 43, 44, 45, 46, 47, 48, 49, 50.

[0020] In some specific embodiments, the 6-phosphate glucose dehydrogenase mutant of the present application is directional coupled to the hapten at a molar ratio of preferably 1:1.

[0021] 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.

[0022] According to the present application, skilled artisans will understand that "hapten" also includes its derivative forms. In order to facilitate coupling with 6-phosphate glucose dehydrogenase, haptens (such as CG) that do not themselves carry a coupling group (e.g., a group reactive with a sulfhydryl group) can be modified to carry a linker to facilitate covalent binding with a sulfhydryl group. Therefore, in the present application, a hapten derivative refers to a hapten that has been modified to carry a sulfhydryl reactive group.

[0023] The hapten is selected from: small molecule drugs (such as antibiotics, psychotropic drugs), hormones, metabolites, sugars, lipids, amino acids, short peptides (molecular weight less than 4000 kDa, or amino acid length not exceeding 50 residues).

[0024] Haptens include, but are not limited to, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides, 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, anti-triiodothyronine, free thyroxine, free triiodothyronine , cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, 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.

[0025] In a specific embodiment, the hapten is glycocholic acid or a derivative thereof. Although glycocholic acid is used as a specific example, it will be understood by those skilled in the art that the technical effect of the present application is not dependent on the specific type of hapten and is applicable to any hapten that can be immunologically detected by competitive methods.

[0026] In a specific embodiment, the hapten is a glycocholic acid derivative having a sulfhydryl reactive group, such as imide, bromoacetyl, vinyl sulfone or aziridine. In a specific embodiment, the hapten is a glycocholic acid derivative, as shown in Formula I:

[0027]

[0028] According to some embodiments, a reagent is provided, which comprises the conjugate of the present application.

[0029] According to some embodiments, provided is a use of the 6-phosphate glucose dehydrogenase mutant of the present application in preparing a detection reagent.

[0030] According to some embodiments, there is provided use of the conjugate of the present application in preparing a detection reagent.

[0031] 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.

[0032] In a specific embodiment, the detection reagent is preferably a reagent based on competition detection.

[0033] According to some embodiments, a glycocholic acid detection kit is provided, comprising:

[0034] - a first reagent, comprising a substrate and a glycocholic acid antibody; the substrate is a substrate of 6-phosphate glucose dehydrogenase;

[0035] - a second reagent, which comprises the conjugate of the present application;

[0036] -optionally, a calibrator comprising 10 mM to 500 mM buffer, 0 mg / L to 40 mg / L glycocholic acid; and

[0037] -Optionally, a quality control product, wherein the quality control product comprises 10 mM to 500 mM buffer and 0 mg / L to 40 mg / L glycocholic acid.

[0038] According to one embodiment, a glycocholic acid detection kit is provided, comprising:

[0039] The first reagent comprises:

[0040] 10mM to 500mM buffer,

[0041] 5mM to 25mM substrate,

[0042] 0.1mg / L to 1mg / L glycocholic acid antibody,

[0043] 10mM to 300mM NaCl,

[0044] 0.1g / L to 5g / L stabilizer,

[0045] 0.1g / L to 5g / L surfactant,

[0046] 0.1g / L to 5g / L preservatives;

[0047] A second reagent comprising:

[0048] 10mM to 500mM buffer,

[0049] The conjugate according to claim 5,

[0050] 0.1g / L to 5g / L stabilizer,

[0051] 0.1g / L to 5g / L surfactant,

[0052] 0.1g / L to 5g / L preservatives.

[0053] In some embodiments, the buffer is selected from one or a combination of the following: 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 100 mM; the pH of the buffer is 6.5 to 7.5, preferably 7.2 or 7.0.

[0054] 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.

[0055] 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.

[0056] In some embodiments, the preservative is selected from one or a combination of the following: azide, MIT, PC-300, thimerosal; and the azide is selected from sodium azide and lithium azide.

[0057] In some embodiments, the substrate comprises: glucose-6-phosphate, β-nicotinamide adenine dinucleotide.

[0058] In some embodiments, the concentration of the buffer is 100 mM.

[0059] In some embodiments, the substrate concentration for the G6PDH enzyme-catalyzed reaction is 5 mM.

[0060] In some embodiments, the concentration of the glycocholic acid antibody is 0.1 mg / L.

[0061] In some embodiments, the concentration of NaCl is 300 mM.

[0062] In some embodiments, the concentration of the stabilizer is 0.5 g / L.

[0063] In some embodiments, the concentration of the surfactant is 0.1 g / L.

[0064] In some embodiments, the concentration of the preservative is 1 g / L.

[0065] According to some embodiments, a method for preparing a conjugate is provided, comprising the steps of:

[0066] 1) providing a hapten or a derivative thereof according to the present application, in particular providing the hapten or a derivative thereof according to the present application in an aprotic solvent (such as but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0067] 2) providing the 6-phosphate glucose dehydrogenase mutant of the present application, 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);

[0068] 3) contacting the 6-phosphate glucose dehydrogenase mutant and the hapten or its derivative at a molar ratio of 1:n for 1 to 4 hours (preferably 2 to 3 hours) at 18° C. to 28° C. to allow the hapten or its derivative to couple with the 6-phosphate glucose dehydrogenase mutant to obtain the conjugate;

[0069] 4) If necessary, the conjugate may be purified, for example, by desalting.

[0070] 43, 44, 45, 46, 47, 48, 49, 50.

[0071] In some specific embodiments, steps 1) and 2) can be interchanged or performed in parallel.

[0072] In some specific embodiments, prior to conjugation, the glucose-6-phosphate dehydrogenase contains a free sulfhydryl group, thereby allowing a 1:1 directed reaction with a hapten or a derivative thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1 .Structure diagram of glycocholic acid.

[0074] Figure 2 .Structure diagram of glycocholic acid derivatives.

[0075] Figure 3A.G6PDH (wild type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides.

[0076] Figure 3B .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0077] Figure 3C .G6PDH(D375C) amino acid sequence (SEQ ID No.3).

[0078] Figure 3D .G6PDH (G426C) amino acid sequence (SEQ ID No. 4). DETAILED DESCRIPTION

[0079] Example

[0080] Example 1. Synthesis of glycocholic acid derivatives

[0081] Add glycocholic acid (1.0 eq), maleimidoethylamine (1.0 g, 1.0 eq), and triethylamine (3.0 eq) into a dry, clean 25 mL two-necked flask;

[0082] Add dimethylformamide (5 mL) and stir until completely dissolved, add dichloroethane (1.25 eq), and stir at 25 °C for 2 h;

[0083] HPLC monitoring until the reaction is complete;

[0084] The above reaction mixture was added to water (25 mL), and ethyl acetate (20 mL × 3) was added for extraction;

[0085] The organic phases were combined, dried over anhydrous Na2SO4, and concentrated under reduced pressure. The resulting oil was purified by column chromatography to give 1.04 g of an off-white powdery solid with a yield of 45% and M+: 602.72.

[0086] The purpose of this embodiment is to make CG carry a group that can bind to the enzyme. The technical effect of this application does not depend on a specific hapten derivative.

[0087] Example 2. Coupling of glycocholic acid derivatives with G6PDH molecules

[0088] According to the G6PDH-glycocholic acid conjugate of the present application, the conjugation is carried out in the following manner: the sulfhydryl reactive group (such as maleimide group) on the glycocholic acid derivative molecule is covalently bonded to the sulfhydryl group on the G6PDH molecule.

[0089] 1. Solution preparation:

[0090] Glycocholic acid derivative solution: 10 mg / ml of the glycocholic acid derivative prepared in Example 1 was dissolved in DMF;

[0091] G6PDH solution: 6.7 mg / mL G6PDH (mutant of the present application or control mutant), 100 mmol PB, 100 mmol NaCl, pH = 8.0;

[0092] Coupling solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2;

[0093] Desalting solution: 100 mM PB / K, 100 mM EDTA, 150 mM NaCl, pH = 7.2.

[0094] 2. Coupling operation: 1.6 ml of G6PDH solution, 6 ml of coupling solution and 0.40 ml of glycocholic acid derivative solution were reacted at room temperature (20 to 25°C) for 4 hours.

[0095] 3. After the reaction system was shaken at room temperature for 4 hours, it was eluted using a desalting column with the desalting solution, and the protein peak was collected. The obtained product was G6PDH-glycocholic acid conjugate.

[0096] Example 3. Preparation of kit

[0097] The following kit for detecting glycocholic acid was prepared, comprising:

[0098] Reagent R1, containing:

[0099] 100 mM PB buffer, pH 7.2

[0100] 15 mM glucose 6-phosphate

[0101] 15 mM β-nicotinamide adenine dinucleotide

[0102] 0.1mg / L glycocholic acid antibody

[0103] 200mM NaCl

[0104] 0.5g / L bovine serum albumin

[0105] 0.1g / L Tween20

[0106] 1g / L sodium azide;

[0107] Reagent R2, including:

[0108] 100 mM PB buffer, pH 7.2

[0109] 0.1mg / L G6PDH-CG conjugate

[0110] 0.5g / L bovine serum albumin

[0111] 0.1g / L Tween 20

[0112] 1g / L sodium azide;

[0113] Calibrator: 100 mM PB buffer, pH 7.2, and 0, 2.5, 5.0, 10, 20, 40 mg / L glycocholic acid (or as needed);

[0114] Quality control: 100 mM PB buffer, pH 7.2, and 1.5, 8.0, 25, and 35 mg / L glycocholic acid (or add as needed).

[0115] Test example

[0116] Reaction time: 10 minutes, including a 4.7-minute incubation time. After adding reagent R2 and incubating for 1 minute, measure the absorbance A1. After another 1-minute incubation, measure the absorbance A2. Calculate ΔA = (A2 - A1) / min. Calculate the glycocholic acid content in the sample using the calibration curve: CG = sample tube absorbance * calibrator concentration / calibrator absorbance.

[0117] The performance of the glycocholic acid detection kit prepared in Example 3 was tested. The main detection performances were total imprecision, repeatability, recovery, linearity, specificity, etc.

[0118] Table 1. Parameters of fully automatic biochemical analyzer

[0119] Detection model Hitachi 7180 Analysis / Time / Point 2-point rate / 10min / 20-24 points R1 / R2 / S 120:40:9 Wavelength (secondary / primary) 405 / 340 Reaction type Increment Calibration type Spine type Calibration points 6 Calibrator concentration 0 / 2.50 / 5.00 / 10.00 / 20.00 / 40.00

[0120] Test Example 1. Calibration of absorbance of glycocholic acid detection kit

[0121] Table 2. Calibrated absorbance of the glycocholic acid detection kit

[0122]

[0123] Test Example 2. Total Imprecision of Glycocholic Acid Test Kit

[0124] Table 3. Total imprecision

[0125]

[0126]

[0127] Test Example 3. Repeatability of the Glycocholic Acid Detection Kit

[0128] Table 4. Repeatability

[0129]

[0130] Detection Example 4. Recovery of Glycocholic Acid Detection Kit

[0131] Table 5. Recycling

[0132]

[0133]

[0134] Test Example 5. Linearity of Glycocholic Acid Detection Kit

[0135] Table 6. Linearity

[0136]

[0137]

[0138] Detection Example 6. Anti-specificity of the Glycocholic Acid Detection Kit

[0139] Table 7. Specificity

[0140] Interferors (40 μg / ml) Reagent of this application (D306C) Control reagent (A45C mutant) Glycodeoxycholic acid 18.61％ 30.20％ Glycochenodeoxycholic acid 1.63％ 37.91％ Chenodeoxycholic acid 0.61％ 16.28％ Ursodeoxycholic acid -0.42％ 6.55％ Sodium cholate 61.21％ 61.90％ Sodium deoxycholate 4.22％ 11.74％

[0141] The reagent of the present application has little or no cross-reaction with the structural analogues of glycocholic acid.

[0142] Test Example 7. Antibody Inhibition Rate

[0143] 1. Principle of Antibody Inhibition Rate Detection

[0144] When the antibody binds to the G6PDH-CG 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.

[0145] 2. Reaction system:

[0146] Table 8. Preparation of antibody inhibition rate detection reagents

[0147]

[0148] Table 9. Antibody inhibition rate test parameters

[0149] Detection model Hitachi 7180 Analysis / Time / Point 2-point rate / 10min / 20-24 points R1 / S 120:20 Wavelength (secondary / primary) 405 / 340 Reaction type Increment

[0150] 3. Results:

[0151] By comparing the absorbance values ​​of the G6PDH-CG conjugate when the antibody is added and when the antibody is not added, the inhibition of the antibody on G6PDH can be obtained.

[0152] Antibody inhibition rate = absorbance change of G6PDH-CG in the presence of antibody / absorbance change of G6PDH-CG in the absence of antibody.

[0153] Compared with previously published mutation sites, the mutants in this application have significantly improved antibody inhibition rates, reaching over 30% and up to 50%, while the inhibition rates of previously commonly used mutation sites (such as A45C and K55C) are only around 20% or even lower.

[0154] Table 10. Antibody inhibition rates of different G6PDH mutants

[0155]

[0156]

[0157] Although not limited to a specific theory, it can be partially explained as follows: compared with the G6PDH mutants in the prior art, the mutation site (i.e., the site where the free thiol group is introduced) in the enzyme mutants of the present application (D306C, D375C, G426C) 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.

[0158] Because the enzyme mutant has a significant improvement in antibody inhibition rate, it can have a clear advantage in absorbance calibration. After the enzyme mutant is coupled with the hapten to form a conjugate into a kit, the reagent has a significant performance improvement in repeatability, total imprecision, linearity, specificity, etc. due to the improvement of the calibration curve.

Claims

1. Use of the conjugate in the preparation of a detection reagent, wherein: The conjugate is formed by coupling the 6-phosphate glucose dehydrogenase mutant and the hapten in a molar ratio of 1:1; Compared to the wild-type 6-phosphate glucose dehydrogenase of Leuconostoc pseudomesenteroides, the 6-phosphate glucose dehydrogenase mutant comprises any one of the following mutations: D306C, D375C, G426C; The hapten is glycocholic acid or a glycocholic acid derivative.

2. The use according to claim 1, wherein the 6-phosphate glucose dehydrogenase mutant is selected from any one of the following sequences: SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No.

4.

3. The method according to claim 1, wherein the detection reagent is an enzyme-linked immunosorbent assay (ELISA) detection reagent.

4. The method according to claim 1, wherein the detection reagent is a chemiluminescence immunoassay detection reagent. The method according to claim 1 , wherein the detection reagent is a homogeneous enzyme immunoassay detection reagent.

6. The method according to claim 1, wherein the detection reagent is a latex enhanced immunoturbidimetric assay.

7. The use according to claim 1, wherein the glycocholic acid derivative is represented by formula I:

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