Use of conjugates in the preparation of detection reagents

Abstract

This application relates to the use of conjugates in the preparation of diagnostic reagents. Specifically, the glucose-6-phosphate dehydrogenase mutant of this application contains one or a combination of mutations selected from the following, compared to wild-type glucose-6-phosphate dehydrogenase: D306C, D375C, and G426C. Diagnostic kits prepared using the glucose-6-phosphate dehydrogenase mutant of this application exhibit high specificity, high sensitivity, ease of operation, short detection time, and accurate quantification, making them suitable for high-throughput detection.

Description

[0001] This application is a divisional application of patent application 202010004879.2, filed on January 3, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of digoxin detection reagent”. 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 digoxin 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 digoxin is shown below:

[0007]

[0008] Digoxin is a cardiac glycoside that has a positive inotropic effect on the heart, slowing the heart rate and inhibiting cardiac conduction. It is used to treat acute and chronic heart failure, including hypertension, valvular heart disease, and congenital heart disease. It is particularly suitable for heart failure accompanied by atrial fibrillation and a rapid ventricular rate.

[0009] Common adverse reactions include: proarrhythmic effects, nausea, vomiting, lower abdominal pain, and abnormal weakness; less common reactions include: blurred vision, central nervous system reactions (such as depression or confusion); rare reactions include: drowsiness, headache, rash, and urticaria (allergic reaction). Among the manifestations of digitalis poisoning, proarrhythmias are the most important, followed by atrioventricular block, paroxysmal or accelerated junctional tachycardia, paroxysmal atrial tachycardia with atrioventricular block, ventricular tachycardia, sinus arrest, and ventricular fibrillation.

[0010] Therefore, monitoring for adverse reactions to this drug should be taken seriously. Furthermore, due to individual differences in drug metabolism, clinical use should incorporate blood drug concentration monitoring to develop a reasonable dosing regimen and minimize the occurrence of adverse reactions.

[0011] Currently known methods for digoxin detection mainly include: high-performance liquid chromatography (HPLC), chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), homogeneous enzyme immunoassay, and latex agglutination turbidimetry. HPLC requires complex sample pretreatment, is complex and time-consuming, and is expensive; chemiluminescent immunoassay reagents are expensive, unsuitable for routine therapeutic drug detection, and hinder widespread adoption. Existing homogeneous enzyme immunoassay and latex agglutination turbidimetry methods are often limited in application due to complex preparation processes and large batch-to-batch variations.

[0012] The prior art CN108593905A describes a digoxin assay kit and its preparation method. However, the prior art method relies on activating the reactive groups of the small molecule drug (digoxin) itself before reacting with an enzyme. Such a coupling method can result in multiple digoxins being linked to the same glucose hexaphosphate dehydrogenase, and the coupling sites are difficult to ensure consistency, making it difficult to guarantee a directional 1:1 reaction between the small molecule drug and the enzyme, leading to large batch-to-batch variations. Summary of the Invention

[0013] 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 digoxin detection kit.

[0014] 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, D375C, and G426C.

[0015] 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.

[0016] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0017] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0018] 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).

[0019] 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.

[0020] 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.

[0021] In some specific implementations, the preferred molar ratio of the glucose-6-phosphate dehydrogenase mutant to the hapten is 1:1.

[0022] 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.

[0023] According to this application, those skilled in the art will understand that "hapten" also includes its derivative forms. To facilitate coupling with glucose-6-phosphate dehydrogenase, haptens that do not inherently possess a coupling group (e.g., a group that reacts with a thiol group) (e.g., digoxigenin) 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.

[0024] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0025] Haptens include, but are not limited to: theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin and digitoxin), phenolic acids, rapamycin, cyclosporine A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, urinary nicotinic acid metabolite cotinine, urinary morphine, urinary monohydroxyphenol derivatives, neuropeptide tyrosine, plasma glycoproline, polyamines, histamine, thyroid-stimulating hormone, prolactin, placental lactogen, growth hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, inverse triiodothyronine, and free thyroxine. Free triiodothyronine, cortisol, urinary 17-hydroxycorticosteroids, urinary 17-ketosteroids, dehydroepiandrosterone and its sulfate, aldosterone, urinary vanillylmandelic acid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17α-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C-peptide, gastrin, plasma prostaglandins, plasma 6-ketoprostaglandin F1α, prostacyclin, adrenaline, catecholamines, norepinephrine, cholecystokinin, natriuretic peptide, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptide, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, serotonin, neuropeptide Y, osteocalcin.

[0026] In a specific implementation plan, the hapten is digoxin or a derivative thereof.

[0027] In a specific implementation, the hapten is a digoxigenin derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

[0028] In a specific implementation scheme, the hapten is a digoxin derivative, as shown in Formula I:

[0029]

[0030] in,

[0031]

[0032] In some implementations, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

[0033] In some specific implementations, the digoxin derivative has the structure shown in Formula I-1:

[0034]

[0035] in,

[0036]

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

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

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

[0040] In the specific implementation plan, the detection reagents are selected from: enzyme-linked immunosorbent assay (ELISA) reagents, chemiluminescent immunoassay (CIA) reagents, homogeneous enzyme immunoassay (HIA) reagents, and latex-enhanced immunoturbidimetric assay (LTIA) reagents.

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

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

[0043] In a specific implementation, the detection device can be prepared in the form of a well plate (e.g., a 96-well plate), for example, the plate is coated with the reagent according to this application.

[0044] In a specific implementation, the detection device can be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with the reagent according to this application.

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

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

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

[0048] - Optionally, the calibrator comprises 10 mM to 500 mM buffer solution, 0 ng / ml to 5 ng / ml digoxigenin (e.g., any value between 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 ng / ml); and

[0049] -Optionally, a quality control product comprising 10 mM to 500 mM buffer solution and 0.7 ng / ml to 4.5 ng / ml (e.g., any value between 0.7, 0.8, 1.5, 1.8, 2, 3, 4, 4.5 ng / ml).

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

[0051] The first reagent comprises:

[0052] 10mM to 500mM buffer solution

[0053] 5mM to 50mM substrate,

[0054] Digoxin antibody from 0.01 μg / ml to 10 μg / ml (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.5, 2, 3, 4, 5 μg / ml).

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

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

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

[0058] The second reagent comprises:

[0059] 10mM to 500mM buffer solution

[0060] 0.01 μg / ml to 10 μg / ml of the conjugates according to this application (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, 1.0 μg / ml).

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

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

[0063] Preservatives ranging from 0.1 g / L to 5 g / L.

[0064] In some embodiments, the buffer is selected from one or a combination of the following: TAPS, glycerol buffer, phosphate buffer, Tris-HCl buffer, citrate-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolpropane buffer; preferably, phosphate buffer; the concentration of the buffer is from 10 mmol / L to 500 mmol / L, preferably from 50 to 100 mM; the pH of the buffer is from 7 to 8.

[0065] 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.

[0066] In some embodiments, the surfactant is selected from one or a combination of the following: Brij23, Brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween20.

[0067] 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, lithium azide, and PC-300.

[0068] In some embodiments, the substrate comprises: 6-phosphoglucose and β-nicotinamide adenine dinucleotide.

[0069] In some specific implementations, the digoxin antibody is derived from: mice, rats, cats, dogs, primates, cattle, horses, sheep, camels, birds, and humans.

[0070] In some specific implementation schemes, the digoxin antibody is selected from: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen-binding fragments.

[0071] According to some implementation schemes, a method for preparing a coupling compound is provided, including the following steps:

[0072] 1) Provide digoxigenin derivatives according to the present application, especially in aprotic solvents (e.g., but not limited to acetonitrile, dimethylformamide, dimethyl sulfoxide);

[0073] 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);

[0074] 3) At 18°C ​​to 28°C, the glucose-6-phosphate dehydrogenase mutant and the digoxigenin derivative are contacted at a molar ratio of digoxigenin derivative to enzyme of 500:1 to 1:500 (preferably 50:1 to 1:50) for 1 hour to 4 hours (1, 1.5, 2, 2.5, 3, 3.5, 4 hours or any value between them, preferably 2 hours to 3 hours) to couple the digoxigenin derivative and the glucose-6-phosphate dehydrogenase mutant, thereby obtaining the conjugate;

[0075] 4) The conjugate may be purified as needed, for example, by desalting.

[0076] In some embodiments, the contact molar ratio of hapten to enzyme in the reaction system is 1:n, where n is from 1 to 500, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500 and any of the above values.

[0077] In some specific implementations, steps 1) and 2) can be interchanged or run in parallel.

[0078] 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 digoxigenin.

[0079] 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

[0080] Figure 1 .G6PDH (wild type) amino acid sequence (SEQ ID No.1); derived from Leuconostoc pseudomesenteroides.

[0081] Figure 2 .G6PDH(D306C) amino acid sequence (SEQ ID No.2).

[0082] Figure 3The amino acid sequence of G6PDH(D375C) (SEQ ID No. 3).

[0083] Figure 4 The amino acid sequence of G6PDH (G426C) (SEQ ID No. 4). Detailed Implementation

[0084] Example

[0085] Example 1. Synthesis of digoxin derivatives

[0086]

[0087] 1. Synthesis of Compound 2

[0088] Dissolve 1.0 g of digoxin in 95% ethanol (80 ml), then add periodic acid (1.0 g) and water (10 ml) solution, and stir at room temperature (18-28 °C) for 1 hour. Filter to remove residue, remove solvent under reduced pressure, and extract with dichloromethane. Dry the organic phase with anhydrous sodium sulfate, and remove solvent under reduced pressure to give compound 2 (white solid, 0.99 g, 99%).

[0089] 2. Synthesis of Compound 4

[0090] Compound 2 (900 mg, 1.16 mmol) was dissolved in 10 mL of dry methanol. Compound 3 (318 mg, 1.0 mmol) was added to the reaction mixture, and the mixture was stirred at room temperature for 5 minutes. Sodium cyanoborohydride (146 mg, 2.32 mmol) was then added, and the mixture was stirred at room temperature for approximately 12 hours. The solvent was removed under reduced pressure, and the mixture was purified directly by column chromatography to give compound 4 (white solid, 585 mg, 66%).

[0091] 3. Synthesis of Compound 5

[0092] Compound 4 was dissolved in 15 mL of dichloromethane and stirred at room temperature for 30 minutes under nitrogen atmosphere. Then, 10 mL of piperidine was added, and the mixture was stirred at room temperature for 2 hours. The solution was removed under reduced pressure, and compound 5 (460 mg, 78%) was purified by column chromatography.

[0093] 4. Synthesis of digoxin derivatives

[0094] Compound 5 (88 mg, 0.11 mmol) and compound 6 (17 mg, 0.11 mmol) were dissolved in DCM (8 mL), and triethylamine (33 mg, 0.33 mmol) was added dropwise, followed by HATU (50 mg, 0.13 mmol). The mixture was stirred at room temperature for 2 h to obtain the digoxin derivative (white solid, 50 mg, 45%).

[0095] 5. Mass spectrometry and nuclear magnetic resonance analysis confirmed that the structure of the digoxin derivative is correct.

[0096] This embodiment enables digoxin to have a group that can bind to enzymes.

[0097] Example 2. Coupling of digoxin derivatives with G6PDH molecules

[0098] I. Coupling Method of This Application

[0099] According to the G6PDH-digoxigenin conjugate of this application, the coupling is carried out in the following manner: the thiol reactive group (such as, but not limited to, maleimide group) on the digoxigenin derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0100] 1. The digoxin derivative prepared in Example 1 was dissolved in N,N-dimethylformamide (10 mg / ml);

[0101] 2. G6PDH solution: G6PDH (e.g., the mutant of this application) is dissolved in 100 mmol PB, 100 mmol NaCl, pH=8.0;

[0102] 3. Add 200 μl of G6PDH solution to 750 μl of buffer solution (0.05 M Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH = 7.2); then add 50 μl of N,N-dimethylformamide solution of digoxin derivative to it;

[0103] 4. The above mixed solution is shaken thoroughly at room temperature (18-28℃) for 2-3 hours to desalt it, and the protein peak is collected. The resulting product is G6PDH-digoxigenin conjugate.

[0104] II. Comparison and Coupling Method (Refer to the method in CN108593905A)

[0105] Accurately weigh 100-300 mg of digoxin and dissolve it in 5-15 mL of anhydrous ethanol;

[0106] Add 5-15 mL of 10-200 mM sodium periodate to the above solution, shake gently, and stir at room temperature for 0.5-2 hours.

[0107] Add 0.5-1 mL of 0.5-2 M ethylene glycol dropwise and stir at room temperature for 5-10 minutes.

[0108] Add the above reaction mixture dropwise to 5-15 mL of a 2-3% G6PDH solution that is being stirred, adjust the pH of the solution to 9.0-9.5, continue stirring the reaction for 0.5-2 hours, and allow the pH of the solution to stabilize.

[0109] Add 100-200 mg of sodium borohydride and stir to reduce for 12-24 hours;

[0110] The G6PDH-digoxigenin conjugate was purified by G-25 gel chromatography column.

[0111] Example 3. Preparation of the reagent kit

[0112] Prepare a kit for detecting digoxin, comprising:

[0113] Reagent R1 contains:

[0114] TAPS buffer 100mM, pH 7.0

[0115] 15mM glucose-6-phosphate

[0116] 15mM β-nicotinamide adenine dinucleotide

[0117] 0.5 μg / ml digoxin antibody (commercially available antibody, no special restrictions)

[0118] 1g / L bovine serum albumin

[0119] 1g / L Brij

[0120] 1 g / L sodium azide;

[0121] Reagent R2 includes:

[0122] Phosphate buffer 200mM, pH 8.0

[0123] 0.1 μg / ml G6PDH-digoxigenin conjugate

[0124] 100mM NaCl

[0125] 1g / L bovine serum albumin

[0126] 1g / L Brij

[0127] 1 g / L sodium azide;

[0128] Calibrator: 20 mM HEPES buffer, and digoxin at concentrations of 0 ng / ml, 0.5 ng / ml, 1 ng / ml, 2 ng / ml, 3 ng / ml, and 5 ng / ml (or add as needed);

[0129] Quality control: 20mM HEPES buffer, and 0.6-0.8ng / ml, 1.6-2.0ng / ml, and 3.8-4.2ng / ml digoxin (or add as needed).

[0130] Assemble the above reagents (optionally including quality control and calibrators) into a digoxin homogeneous enzyme immunoassay kit.

[0131] Detection example

[0132] The principle of homogeneous enzyme immunoassay: In a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6PDH-digoxigenin) and unlabeled antigen (digoxigenin) compete for binding with a quantitative amount of antibody (digoxigenin antibody). The more the antibody binds to the unlabeled antigen, the more activity of the enzyme-labeled antigen is released, and the more the enzyme catalyzes the generation of NADH from the substrate NAD+.

[0133] The digoxin content in a liquid can be calculated by detecting the change in absorbance of NADH at a wavelength of 340 nm.

[0134] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0135]

[0136]

[0137] Example 1. Performance of the reagent kit of this application

[0138] 1. Calibration Experiment

[0139] Table 2. Calibration absorbance of the digoxin detection kit

[0140]

[0141] 2. Precision Experiment

[0142] Table 3. Total Imprecision

[0143]

[0144] 3. Repeatability

[0145] Table 4. Repeatability

[0146]

[0147]

[0148] 4. Recycling Test

[0149] Table 5. Recycling Data

[0150]

[0151] 5. Linear Experiment

[0152] Table 6. Linear

[0153]

[0154]

[0155] Test Example 2. Airborne Stability

[0156] The absorbance of the reagent (D375C mutant) accelerated at 37℃ for 7 days decreased by less than 10%, while the absorbance of the control reagent decreased significantly after 7 days of acceleration at 37℃.

[0157] Table 7. Accelerated stability at 37℃

[0158]

[0159] Example 3. Antibody inhibition rate

[0160] 1. Detection principle of antibody inhibition rate

[0161] When the antibody binds to the G6PDH-digoxigenin 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.

[0162] 2. Reaction system

[0163] Table 8. Preparation of reagents for detecting antibody inhibition rate

[0164]

[0165] 3. Results

[0166] By comparing the absorbance values ​​of the G6PDH-digoxigenin conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0167] Antibody inhibition rate = (Change in absorbance of G6PDH-digoxigenin conjugate with antibody) / (Change in absorbance of G6PDH-digoxigenin without antibody) × 100%.

[0168] Compared to the published mutation site (A45C), the mutant in this application shows a significant improvement in enzyme activity retention, reaching over 40% (G426C: 40%; D375C: 49%), and up to 62% (D306C). When the published mutation sites (e.g., A45C, K55C) were prepared into G6PDH-digoxigenin conjugates using the method described in this application, the inhibition rates were only 33% and 40%, respectively.

[0169] 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.

[0170] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the reagent kit formulated by conjugating the enzyme mutant with digoxigenin shows significant performance improvements in batch-to-batch coefficient of variation, linearity, repeatability, and stability.

Claims

1. The uses of conjugates in the preparation of detection reagents, including: The detection reagent is a homogeneous enzyme immunoassay reagent for digoxin; The conjugate is formed by conjugating a glucose-6-phosphate dehydrogenase mutant with a digoxigenin derivative in a molar ratio of 1:

1. The digoxin derivative is shown in Formula I-1: in, 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.

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