Use of conjugates in the preparation of detection reagents

Abstract

The present application relates to the use of conjugates in the preparation of detection reagents. 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 to 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 the patent application filed on January 7, 2020, entitled “6-phosphate dehydrogenase mutant and its use in the preparation of methotrexate detection reagent” (application number 202010013644X). 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 methotrexate 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.

[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 methotrexate is shown below:

[0007]

[0008] Methotrexate, also known as aminomethylfolic acid, is an orange-yellow crystalline powder with a melting point of 185 to 204°C. It is stable in air at room temperature, readily soluble in dilute alkali, acid, or alkali metal carbonate solutions, slightly soluble in dilute hydrochloric acid, and almost insoluble in water, ethanol, chloroform, and ether.

[0009] Methotrexate is an antifolate-based antitumor drug that primarily inhibits the synthesis of dihydrofolate reductase, thereby suppressing tumor cell growth and proliferation. Its main functions include systemic treatment of choriocarcinoma, malignant hydatidiform mole, various types of acute leukemia, breast cancer, lung cancer, head and neck cancer, gastrointestinal cancer, cervical cancer, and malignant lymphoma. Arterial infusion via catheterization also shows good efficacy against head and neck cancer and liver cancer. Combining methotrexate with biologics such as adalimumab can more effectively alleviate symptoms in patients with rheumatoid arthritis, slow the progression of joint damage, and improve bodily function. It can also be injected locally into the gestational sac of ectopic pregnancies to relieve bleeding.

[0010] Methotrexate is a primary treatment for leukemia, but after Gubner et al. reported its effectiveness in treating chronic rheumatoid arthritis (RA) in 1951, it gained attention as a treatment for RA and is mainly used in Europe and America. Methotrexate is a prescription drug and must be prescribed by a doctor. High doses can cause osteoporosis, joint pain, eye irritation, and diabetes. It may also cause anovulation and reduced sperm count, leading to decreased fertility. Like other folic acid antagonists, methotrexate has teratogenic effects and can cause stillbirth.

[0011] For the reasons mentioned above, timely monitoring of methotrexate blood concentration is necessary during treatment. This is an effective way to assist clinical treatment, improve treatment efficacy, and reduce toxicity risks.

[0012] Currently known methods for detecting methotrexate include high-performance liquid chromatography (HPLC), chemiluminescent immunoassay, and enzyme-linked immunosorbent assay (ELISA). HPLC can separate the drug from its metabolites and endogenous substances, exhibiting high specificity and serving as the gold standard for detecting MTX plasma concentrations. However, this method requires complex pretreatment and a long assay time, making it unsuitable for rapid detection of large samples. Immunoassays are somewhat affected by cross-reactivity, but their speed and ease of operation have gradually made them a primary method for therapeutic drug monitoring. Chemiluminescent immunoassay reagents are expensive, unsuitable for routine therapeutic drug detection, and hinder widespread adoption.

[0013] Prior art CN104569373A describes a homogeneous enzyme immunoassay reagent for methotrexate and its preparation and detection methods, which discloses the preparation method of glucose-6-phosphate dehydrogenase and methotrexate conjugate:

[0014] a) Prepare a buffer solution by weighing 1.09 g / L potassium dihydrogen phosphate, 1.70 g / L disodium hydrogen phosphate, and 8.5 g / L sodium chloride, and adjusting the pH to 7.4;

[0015] b) Weigh 3 mg of glucose-6-phosphate dehydrogenase and dissolve it in 3 mL of the solution obtained in step a above at room temperature to prepare a glucose-6-phosphate dehydrogenase solution.

[0016] c) Weigh 3 mg of the methotrexate derivative and dissolve it in 300 μL of the solution obtained in step a above at room temperature to prepare a methotrexate derivative solution: The methotrexate derivative is shown below:

[0017]

[0018] d) Mix the solutions obtained in steps b and c above and stir at 2-8°C for 1 hour to allow glucose-6-phosphate dehydrogenase to be linked to the terminal carboxyl group of methotrexate.

[0019] e) Place the mixed solution obtained in step d above into the solution obtained in step a above for dialysis purification to obtain glucose-6-phosphate dehydrogenase-methotrene conjugate.

[0020] The aforementioned prior art only indicates that the enzyme is linked to the terminal carboxyl group of methotrexate. However, there are many groups on the enzyme that can bind to the carboxyl group (such as amino groups), so it is difficult to guarantee directional coupling. Therefore, the problems of inconsistent results and large batch-to-batch differences in the obtained conjugates still exist. Summary of the Invention

[0021] 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 methotrexate detection kit.

[0022] According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. Unlike the glucose-6-phosphate dehydrogenase mutant in the previously published patent 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.

[0023] 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.

[0024] According to some embodiments, a polynucleotide is provided that encodes the glucose-6-phosphate dehydrogenase mutant of this application.

[0025] According to some implementation schemes, an expression vector is provided that contains the polynucleotides of this application.

[0026] 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).

[0027] 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 in a molar ratio of 1:1.

[0028] 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.

[0029] 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., methotrexate) 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.

[0030] Haptens are selected from: small molecule drugs (such as antibiotics and psychotropic drugs), hormones, metabolites, sugars, lipids, and amino acids.

[0031] Haptens include, but are not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxin and digitoxin), phenolic acids, rapamycin, cyclosporine A, methotrexate, 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 glycosaminoglycans, 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, transtriiodothyronine, and other similar substances. Levothyroxine, 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.

[0032] In a specific implementation plan, the hapten is methotrexate or a derivative thereof.

[0033] In a specific implementation, the hapten is a methotrexate derivative with a thiol reactive group, such as lemiimide, bromoacetyl, vinyl sulfone, or aziridine.

[0034] In a specific implementation scheme, the hapten is a methotrexate derivative, as shown in Formula I:

[0035]

[0036] 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.

[0037] In a specific implementation scheme, the methotrexate derivative is shown in Formula II:

[0038]

[0039] According to some embodiments, a reagent is provided that comprises the conjugate of this application.

[0040] According to some implementation schemes, the use of the glucose-6-phosphate dehydrogenase mutant of this application in the preparation of methotrexate detection reagents is provided.

[0041] According to some implementation schemes, the use of the conjugates of this application in the preparation of methotrexate detection reagents is provided.

[0042] 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.

[0043] In a specific implementation plan, the detection reagent is preferably a reagent based on a competitive detection method.

[0044] According to some implementation schemes, the use of the conjugate of this application in the preparation of a methotrexate detection device is provided.

[0045] 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.

[0046] 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.

[0047] According to some implementation schemes, a methotrexate detection kit is provided, comprising:

[0048] - First reagent, comprising a substrate, a buffer solution, and a methotrexate antibody; the substrate is a substrate of glucose-6-phosphate dehydrogenase;

[0049] - A second reagent, comprising the conjugate and buffer solution of this application;

[0050] - Optionally, the calibrator comprises 10 mM to 500 mM buffer, 0 μM to 2.0 μM methotrexate; and

[0051] - Optionally, a quality control sample comprising 10 mM to 500 mM buffer solution and 0.2 μM to 2.0 μM methotrexate.

[0052] According to one embodiment, a methotrexate detection kit is provided, comprising:

[0053] The first reagent comprises:

[0054] 10mM to 500mM buffer solution

[0055] 5mM to 50mM substrate,

[0056] 0.05% to 0.5% w / v, preferably 0.2% to 0.5% w / v methotrexate antibody; 0.05% to 0.5% w / v, preferably 0.05% to 0.1% w / v stabilizer.

[0057] 0.05% to 1% w / v, preferably 0.5% to 1% w / v sodium chloride,

[0058] 0.05% to 0.5% w / v, preferably 0.05% to 0.1% w / v preservative;

[0059] The second reagent comprises:

[0060] 10mM to 500mM buffer solution

[0061] 0.1% to 0.5% w / v of the conjugate according to this application,

[0062] 0.05% to 0.5% w / v, preferably 0.05% to 0.1% w / v stabilizer.

[0063] 0.05% to 1% w / v, preferably 0.5% to 1% w / v sodium chloride,

[0064] 0.05% to 0.5% w / v, preferably 0.05% to 0.1% w / v preservative;

[0065] 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 200 mM; the pH of the buffer is from 7 to 8.4.

[0066] In some embodiments, the stabilizer is selected from one or a combination of the following: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, and polyethylene glycol 8000; preferably bovine serum albumin.

[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 and lithium azide.

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

[0069] In some specific implementation schemes, the methotrexate antibody is derived from: rabbits, mice, rats, goats, sheep, cats, guinea pigs, dogs, primates, cattle, horses, camels, birds, and humans.

[0070] In some specific implementation schemes, the methotrexate 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 a methotrexate derivative 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 methotrexate derivative and the glucose-6-phosphate dehydrogenase mutant are contacted at a molar ratio of n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) to couple the methotrexate 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 enzyme to hapten in the reaction system is 1:n, where n is from 0.1 to 500, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 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 the above values. In one specific implementation, the contact molar ratio of enzyme to hapten in the reaction system is 5:1 to 1:5.

[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 methotrexate.

[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 3 The 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 Methotrexate Derivatives

[0086]

[0087] Add methotrexate (100 mg, 0.22 mmol) to a round-bottom flask and dissolve it in dry N,N-dimethylformamide (10 mL). Add 4-maleimide butyric acid (39 mg, 0.22 mmol) and triethylamine (44 mg, 0.44 mmol). Stir under nitrogen protection until completely dissolved.

[0088] HOAt (45 mg, 0.33 mmol) was added to the reaction system and stirred until completely dissolved. HATU (125 mg, 0.33 mmol) was then added, and the mixture was stirred at room temperature (18-28 °C, preferably 20-25 °C) for approximately 4 hours. The reaction was then monitored by TLC. After the reaction was complete, the product was directly purified using a preparative plate (MeOH / DCM = 5:3) to finally obtain the methotrexate derivative (45 mg, yield 35%).

[0089] The product structure was confirmed using conventional methods. In this embodiment, methotrexate contains a group that can bind to an enzyme.

[0090] Example 2. Coupling of methotrexate derivatives with G6PDH molecules

[0091] I. Coupling Method of This Application

[0092] According to the G6PDH-methopterin 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 methopterin derivative molecule is covalently bonded to the thiol group on the G6PDH molecule.

[0093] 1. The methotrexate derivative prepared in Example 1 was dissolved in N,N-dimethylformamide (10 mM);

[0094] 2. G6PDH solution: G6PDH (the mutant of this application or the mutant of the prior art) is dissolved in 0.2M phosphate buffer, pH 8.0 (6.4mM enzyme);

[0095] 3. Add 200 μl of glucose-6-phosphate dehydrogenase mutant solution to 750 μL of buffer solution (0.05 mol / L Na2HPO4, 150 mM NaCl, 10 mM EDTA, 0.1% NaN3, pH=7.2), and then add 50 μl of methotrexate derivative solution.

[0096] 4. The above mixed solution is shaken thoroughly at room temperature (18-28℃, preferably 20 to 25℃) for 2-3 hours to desalt it, and the resulting product is G6PDH-methotrexate conjugate (concentration 0.1mM-2.0mM).

[0097] II. Comparison Coupling Method A

[0098] G6PDH-methotrexate conjugate was prepared according to the method disclosed in Example 4 of CN104569373A.

[0099] III. Contrast Coupling Method B (relying on the activation of the self-contained group of methotrexate)

[0100] 1. Dissolve 2.38 mL of glucose-6-phosphate dehydrogenase in 12 mL of Tris buffer, then add 225 mg of reduced nicotinamide adenine dinucleotide (NADH), 135 mg of glucose-6-phosphate, and 2.25 mL of dimethyl sulfoxide sequentially; the pH of the Tris buffer is 9.0, and the concentrations of each component are: 0.05 mol / L Tris, 3.3 mM magnesium chloride, and 145.4 mM sodium chloride.

[0101] 2. Activation of methotrexate derivatives: 10 mg of methotrexate derivatives were dissolved in 420 μL of dimethyl sulfoxide and 180 μL of dimethylformamide, and 6 μL of tributylamine and 350 μL of isobutyl chloroformate were added. The mixture was stirred at 2-8 °C for 30 minutes.

[0102] 3. Mix the solutions obtained in steps 1 and 2, stir at 2-8°C for 12-16 hours, and purify the conjugated enzyme-labeled antigen by G-25 gel chromatography to obtain glucose-6-phosphate dehydrogenase-labeled methotrexate derivatives.

[0103] Example 3. Preparation of the reagent kit

[0104] Prepare the following kit for the detection of methotrexate, comprising:

[0105] 1. Preparation of the first reagent:

[0106]

[0107] 2. Preparation of the second reagent:

[0108]

[0109] 3. Calibrator: 20mM HEPES buffer, and 0.2-0.8μM, 0.9-1.2μM, and 1.3-2.0μM methotrexate (or add as needed);

[0110] 4. Quality control materials: 20mM HEPES buffer, and 0μM, 0.13μM, 0.25μM, 0.50μM, 1.00μM, and 2.00μM methotrexate (or add as needed).

[0111] 5. Assemble the above reagents (optionally including quality control samples and calibrators) into a test kit.

[0112] Detection example

[0113] In a homogeneous reaction system, methotrexate and the G6PDH-methotrexate conjugate in the sample simultaneously compete for binding sites to anti-methotrexate antibodies. Since enzyme activity decreases after antibody binding to the conjugate, the more free methotrexate in the sample, the more antibody sites compete for binding, and the less antibody binds to the enzyme conjugate. The unbound enzyme conjugate catalyzes the oxidation of β-nicotinamide adenine dinucleotide (NAD). + Methotrexate is converted into β-nicotinamide adenine dinucleotide (NADH). The concentration of methotrexate in the sample is directly proportional to the amount of NADH generated. The concentration of methotrexate in the sample can be obtained by observing the change in absorbance.

[0114] Table 1. Parameters of Fully Automated Biochemical Analyzer

[0115]

[0116]

[0117] Example 1. Performance of the reagent kit of this application

[0118] 1. Calibration absorbance

[0119] Methotrexate calibrator was dissolved in a buffer solution (0.9% NaCl, 0.1% NaN3) to prepare calibrators of six concentrations. The calibration working volume was 2-10 μL, then 100-200 μL of the first reagent and 50-100 μL of the second reagent were added. Using the rate method, the absorbance change rate at a specific reading point was detected at a dominant wavelength of 340 nm and a secondary wavelength of 405 nm, and a calibration curve was plotted. The establishment and optimization of the calibration curve in this application were performed on a Hitachi 7180, but it has been tested and found to be usable on other mainstream models (AU680, Abbott C16000, etc.).

[0120] 2. Precision Experiment

[0121] Using the calibration curves established above, high, medium, and low quality control products and clinical samples were measured.

[0122] Table 2. Precision

[0123]

[0124]

[0125] 3. Linear

[0126] Table 3. Linear

[0127]

[0128]

[0129] 4. Airborne stability (D375C mutant)

[0130] Three quality control samples of high, medium and low concentrations were selected, and the tests were performed three times a day or every other day. The data showed that the calibration cycle of the reagent kit of this application could be stable for more than two weeks. After the reagent was opened, it was placed on the machine, and the test showed that the on-board stability exceeded 40 days. The data are shown in Table 6 (where 40 days* indicates the test data after 40 days of on-board recalibration).

[0131] 5. Drug Interference Experiment

[0132] Twenty commonly used compounds and drugs were selected. At a methotrexate concentration of 10 μM, the following concentrations of compounds did not significantly interfere with the detection of the D375C mutant in the kit of this application.

[0133] Table 4. Drug Interference Experiments

[0134]

[0135]

[0136] Example 2. Antibody inhibition rate in conjugates

[0137] 1. Detection principle of antibody inhibition rate

[0138] When the antibody binds to the G6PDH-methotrexate 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.

[0139] 2. Reaction system

[0140] Table 5. Preparation of reagents for detecting antibody inhibition rate

[0141]

[0142] 3. Results

[0143] By comparing the absorbance values ​​of the G6PDH-methotrexate conjugate with and without the addition of antibody, the inhibitory effect of the antibody on G6PDH can be obtained.

[0144] Compared to conjugates prepared from previously published mutant sites (A45C, K55C), the enzyme mutant of this application exhibits a significantly improved antibody inhibition rate, reaching over 44.6% (G426C: 44.6%; D375C: 51%), with a maximum of 60.3% (D306C). Previously published mutant sites (e.g., A45C, K55C) showed inhibition rates ranging from 39.7% to 42.2%.

[0145] 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 sulfhydryl group) in the enzyme mutant of this application is the location where it couples with a hapten (such as a hormone or small molecule drug). When the hapten binds to a hapten-specific antibody at this site, the resulting steric hindrance significantly affects the activity of the G6PDH enzyme, while 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 sufficient exposure of the hapten epitope.

[0146] Because the enzyme mutant exhibits a significant increase in antibody inhibition rate, the formulation of a kit by conjugating the enzyme mutant with methotrexate resulted in a marked improvement in performance in terms of batch-to-batch coefficient of variation, linearity, and specificity.

[0147]

Claims

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

1. The methotrexate derivative is shown in Formula II: Formula II; 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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