CN116298330A - Use of conjugates in the preparation of detection reagents - Google Patents

Abstract

The present application relates to the use of conjugates in the preparation of detection reagents. Specifically, the mutant glucose 6-phosphate dehydrogenase of the present application comprises one mutation or a combination thereof selected from the group consisting of: d306C, D375C, G426C. The detection kit prepared by using the glucose 6-phosphate dehydrogenase mutant has the advantages of strong specificity, high sensitivity, convenient operation, short detection time, accurate quantification and suitability for high-throughput detection.

Description

Use of conjugates in the preparation of detection reagents

The application is a divisional application of patent application No. 202010013644X of 6-phosphoglucose dehydrogenase mutant and application of mutant in preparation of methotrexate detection reagent, which is filed on 1/7/2020.

Technical Field

The application relates to the field of biological detection, in particular to mutant enzyme glucose 6-phosphate dehydrogenase (G6 PDH for short) and application thereof in a methotrexate detection kit.

Background

Hapten, some small molecule substances (molecular weight less than 4000 Da) alone are not able to induce an immune response, i.e. are not immunogenic, but are immunogenic when crosslinked or conjugated to a carrier such as a macromolecular protein or non-antigenic polylysine, inducing an immune response. These small molecule substances can bind to the response effect products, are antigenic, are only immunoreactive, are not immunogenic, and are also called incomplete antigens.

Hapten can bind to the corresponding antibody to generate antigen-antibody reaction, and antigen which can not independently excite human or animal body to generate antibody can not be generated. It is only immunoreactive, not immunogenic, also known as incomplete antigen. Most polysaccharides, lipids, hormones and small molecule drugs belong to the hapten group. If hapten is chemically bound to a protein molecule (carrier), new immunogenicity is obtained and the animal is stimulated to produce the corresponding antibody.

Small molecule antigens or haptens, which lack two or more sites available for sandwich methods, cannot be assayed by the double antibody sandwich method, and are often in competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with the solid phase antibody. The more the antigen content in the specimen, the less the enzyme-labeled antigen is bound on the solid phase, and the lighter the color development. ELISA assay for small molecule hormones, drugs and the like is commonly used.

Methotrexate (Methotrexate) has the following structural formula:

methotrexate is also known as aminomethylfolic acid, methotrexate, orange yellow crystalline powder, and has a melting point of 185-204 ℃ and is stable in air exposure at normal temperature, is easily dissolved in dilute alkali, acid or alkali metal carbonate solution, is slightly dissolved in dilute hydrochloric acid, and is almost insoluble in water, ethanol, chloroform and diethyl ether.

Methotrexate is antifolate antitumor drug, and can inhibit synthesis of tumor cells by inhibiting dihydrofolate reductase, thereby inhibiting growth and proliferation of tumor cells. The medicine is mainly used for treating chorionic epithelial cancer, malignant grape embryo, various acute leukemia, breast cancer, lung cancer, head and neck cancer, digestive tract cancer, cervical cancer, malignant lymphoma and the like. The arterial cannula is used for perfusion to treat head and neck cancer and liver cancer, and the biological agents such as methotrexate and adalimumab are used together, so that the disease symptoms of patients suffering from joint rheumatism can be effectively relieved, the progress of joint injury is slowed down, and the body functions can be improved. Can also be used for relieving hemorrhage by local embryo sac injection in ectopic pregnancy.

Methotrexate is a major therapeutic agent for leukemia, but in 1951 gudner et al proposed that aminopterin is effective for chronic RA, and is attracting attention as a therapeutic agent for RA, and is mainly used in europe and america. Methotrexate is a prescribed drug that must be prescribed by a physician and high doses can cause osteoporosis, joint pain, eye irritation, and diabetes, as well as may cause egg cell deficiency and oligospermia, hypofertility. Like other folic acid antagonists, methotrexate also has teratogenic effects and can cause stillbirth.

For the above reasons, methotrexate plasma concentration monitoring needs to be performed in time during the treatment process, and is an effective way for assisting clinical treatment, improving treatment effect and reducing toxicity risk.

The currently known methotrexate detection methods mainly comprise: high Performance Liquid Chromatography (HPLC), luminescence immunity, enzyme-linked immunosorbent assay (ELISA), etc. The high performance liquid chromatography can separate the drug from the metabolite and endogenous substances, has the characteristic of strong specificity, is a gold standard for detecting MTX plasma concentration, but the method requires a complex pretreatment process and long measurement time, and is not suitable for rapid detection of large samples. Immunological methods are affected by cross-reactions to some extent, but they have become the main method of therapeutic drug monitoring by virtue of their rapid and easy operation. The luminous immunoassay method has the disadvantages of high reagent cost, inapplicability to detection of conventional therapeutic drugs and inapplicability to large-scale popularization.

The prior art CN104569373a describes a methotrexate homogeneous enzyme immunoassay reagent and a preparation and detection method thereof, wherein the preparation method of the conjugate of glucose-6-phosphate dehydrogenase and methotrexate is disclosed:

a) Preparing a buffer solution, weighing 1.09g/L potassium dihydrogen phosphate, 1.70g/L disodium hydrogen phosphate and 8.5g/L sodium chloride, and regulating the pH to 7.4;

b) Weighing 3mg of glucose 6-phosphate dehydrogenase, and dissolving the glucose 6-phosphate dehydrogenase in 3mL of the solution obtained in the step a at room temperature to prepare a glucose 6-phosphate dehydrogenase solution;

c) 3mg of the methotrexate derivative was weighed and dissolved in 300. Mu.L of the solution obtained in the step a at room temperature to prepare a methotrexate derivative solution: methotrexate derivatives are shown below:

d) Mixing the solutions obtained in the steps b and c, and stirring at 2-8 ℃ for 1 hour to enable the glucose-6-phosphate dehydrogenase to be connected with the terminal carboxyl of the methotrexate;

e) And d, placing the mixed solution obtained in the step d into the solution obtained in the step a for dialysis and purification to obtain the glucose 6-phosphate dehydrogenase-methotrexate conjugate.

The prior art only indicates that the enzyme is connected with the terminal carboxyl of methotrexate, however, a plurality of groups (such as amino groups) which can be combined with the carboxyl exist on the enzyme, so that directional coupling is difficult to ensure, and the problems of low consistency of the obtained conjugate and large difference among kit batches still exist.

Disclosure of Invention

In view of the needs in the art, the present application provides a novel glucose 6-phosphate dehydrogenase mutant and its use in preparing a methotrexate detection kit.

According to some embodiments, a glucose 6-phosphate dehydrogenase mutant is provided. Unlike the glucose 6 phosphate dehydrogenase mutant of the prior published patent US006090567A (Homogeneous immunoassays using mutant glucose-6-phosphate dehydrogenases), the glucose 6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: d306C, D375C, G426C.

According to some embodiments, there is provided a glucose 6-phosphate dehydrogenase mutant, the glucose 6-phosphate dehydrogenase mutant being represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No.4.

According to some embodiments, a polynucleotide encoding a glucose 6-phosphate dehydrogenase mutant of the present application is provided.

According to some embodiments, there is provided an expression vector comprising a polynucleotide of the present application.

According to some embodiments, a host cell is provided comprising an expression vector of the present application. The host cell may be prokaryotic (e.g., bacteria) or eukaryotic (e.g., yeast).

According to some embodiments, there is provided a conjugate which is a glucose 6-phosphate dehydrogenase mutant of the present application and a hapten in a molar ratio of 1:1 is coupled.

In some specific embodiments, the hapten has a molecular weight of 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, 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.

In light of the present application, the skilled artisan will appreciate that "hapten" also includes the form of its derivative. In order to facilitate the coupling with glucose-6-phosphate dehydrogenase, haptens (e.g.methotrexate) that do not themselves carry a coupling group (e.g.a group that reacts with a thiol group) may be engineered to carry a linker for covalent binding to the thiol group. Thus, in the present application, hapten derivatives refer to haptens engineered to bear a thiol-reactive group.

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

Hapten such as, but not limited to: vancomycin, theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including digoxin, digitoxin), zymophenolic acid, lei Paming, cyclosporin A, methotrexate, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, the metabolite cotinine of uronikotin, uromorphine, derivatives of uromonohydric phenol, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyrotropic hormone, prolactin, placental prolactin, 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-hydroxycortic, urinary 17-ketosteroid, dehydroepiandrosterone and sulfate, aldosterone, urovanilloid, plasma renin, angiotensin, erythropoietin, testosterone, dihydrotestosterone, androstenedione, 17 alpha-hydroxyprogesterone, estrone, estriol, estradiol, progesterone, human chorionic gonadotropin, insulin, proinsulin, C peptide, gastrin, plasma prostaglandin, plasma 6-keto prostaglandin f1α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, natriuretic acid, cyclic adenosine monophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, substance P, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptides Y, osteocalcin.

In a specific embodiment, the hapten is methotrexate or a derivative thereof.

In a specific embodiment, the hapten is a methotrexate derivative with a sulfhydryl reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

In a specific embodiment, the hapten is a methotrexate derivative, as shown in formula I:

in some embodiments, m is an integer from 1 to 10, preferably an integer from 1 to 5, such as 1, 2, 3, 4, 5.

In a specific embodiment, the methotrexate derivative is represented by formula II:

according to some embodiments, there is provided an agent comprising a conjugate of the present application.

According to some embodiments, there is provided the use of a glucose 6-phosphate dehydrogenase mutant of the present application for the preparation of a methotrexate detection reagent.

According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a methotrexate detection reagent.

In specific embodiments, the detection reagent is selected from the group consisting of: ELISA detection reagent, chemiluminescent detection reagent, homogeneous ELISA detection reagent and latex enhanced turbidimetry detection reagent.

In a specific embodiment, the detection reagent is preferably a reagent for competition-based detection.

According to some embodiments, there is provided the use of a conjugate of the present application in the preparation of a methotrexate detection device.

In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., 96-well plate), such as a plate coated with reagents according to the present application.

In particular embodiments, the detection device may be prepared in the form of particles (e.g., latex, magnetic beads), such as particles coated with a reagent according to the present application.

According to some embodiments, there is provided a methotrexate detection kit comprising:

-a first agent comprising a substrate, a buffer and a methotrexate antibody; the substrate is a substrate for glucose-6-phosphate dehydrogenase;

-a second agent comprising a conjugate of the present application and a buffer;

-optionally, a calibrator comprising 10mM to 500mM buffer, 0 μm to 2.0 μm methotrexate; and

-optionally, a quality control comprising 10mM to 500mM buffer, 0.2 μm to 2.0 μm methotrexate.

According to one embodiment, there is provided a methotrexate detection kit comprising:

a first reagent comprising:

10mM to 500mM buffer,

5mM to 50mM substrate,

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,

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

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

a second reagent comprising:

10mM to 500mM buffer,

0.1% to 0.5% w/v of a conjugate according to the present application,

0.05 to 0.5% w/v, preferably 0.05 to 0.1% w/v, of a stabilizer,

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

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

in some embodiments, the buffer is selected from one or a combination of the following: TAPS, tromethamine buffer, phosphate buffer, tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, and trimethylol methane buffer; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 50 to 200mM; the pH of the buffer is 7 to 8.4.

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; bovine serum albumin is preferred.

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

In some embodiments, the substrate comprises: glucose-6-phosphate, beta-nicotinamide adenine dinucleotide.

In some specific embodiments, the methotrexate antibody is derived from: rabbits, mice, rats, goats, sheep, cats, guinea pigs, dogs, primates, cows, horses, camelids, birds, humans.

In some specific embodiments, the methotrexate antibody is selected from the group consisting of: monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, and antigen binding fragments.

According to some embodiments, there is provided a method of preparing a conjugate comprising the steps of:

1) Providing a methotrexate derivative according to the present application, in particular in an aprotic solvent (e.g. but not limited to acetonitrile, dimethylformamide, dimethylsulfoxide);

2) Providing a glucose 6-phosphate dehydrogenase mutant, preferably in a buffer (which provides a reaction environment such as, but not limited to PBS, tris, TAPS, TAPSO, said buffer having a pH of 6.0 to 8.0);

3) Contacting the methotrexate derivative and the glucose 6-phosphate dehydrogenase mutant at a molar ratio n:1 for 1 hour to 4 hours (preferably 2 hours to 3 hours) at 18 ℃ to 28 ℃ such that the methotrexate derivative and the glucose 6-phosphate dehydrogenase mutant are coupled to obtain the seed conjugate;

4) The seed conjugate is optionally purified, e.g., desalted, etc., as desired.

In some embodiments, the contacting molar ratio of enzyme to hapten in the reaction system is 1: n, where n is 0.1 to 500, e.g., 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 ranges between any of the foregoing values thereof. In a specific embodiment, the contacting molar ratio of enzyme to hapten in the reaction system is 5:1 to 1:5.

in some specific embodiments, steps 1) and 2) can be interchanged or in parallel.

In some specific embodiments, the glucose 6-phosphate dehydrogenase comprises one or more free sulfhydryl groups prior to coupling, thereby allowing for a directed reaction with methotrexate.

Wild-type glucose 6-phosphate dehydrogenase does not contain a free thiol group, and thus in some specific embodiments, the glucose 6-phosphate dehydrogenase is genetically engineered to have an amino acid mutation at a particular site (306, 375, or 426) to a cysteine, thereby carrying a free thiol group.

Drawings

FIG. 1G 6PDH (wild type) amino acid sequence (SEQ ID No. 1); is derived from Leuconostoc pseudoenteroides Leuconostoc pseudomesenteroides.

FIG. 2G 6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3G 6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 4G 6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

EXAMPLE 1 Synthesis of methotrexate derivatives

To a round bottom flask was added methotrexate (100 mg,0.22 mmol), dissolved in dry N, N-dimethylformamide (10 mL), 4-maleimidobutyric acid (39 mg,0.22 mmol) was added, triethylamine (44 mg,0.44 mmol) was added, and stirred under nitrogen until all dissolved.

HOAt (45 mg,0.33 mmol) was added to the reaction, stirred until all dissolved, HATU (125 mg,0.33 mmol) was added, stirred at room temperature (18-28 ℃ C., preferably 20-25 ℃ C.) for about 4h, and TLC detection. Purification by prep plate (MeOH/dcm=5:3) was performed directly after the reaction, and methotrexate derivative was finally obtained (45 mg, 35% yield).

The structure of the product was confirmed by a conventional method. This example provides methotrexate with a group that binds to enzymes.

EXAMPLE 2 coupling of methotrexate derivatives with G6PDH molecules

1. Coupling methods of the present application

The G6 PDH-methotrexate conjugate according to the present application is coupled as follows: thiol-reactive groups (such as but not limited to maleimide groups) on methotrexate derivative molecules are covalently bound to thiol groups on G6PDH molecules.

1. The methotrexate derivative prepared in example 1 was dissolved in N, N-dimethylformamide (10 mM);

g6pdh solution: g6PDH (mutant of the present application or prior art mutant) was dissolved in 0.2M phosphate buffer, pH 8.0 (6.4 mM enzyme);

3. 200. Mu.l of glucose 6 phosphate dehydrogenase mutant solution was added to 750. Mu.l of buffer solution (0.05 mol/L Na) ₂ HPO ₄ 、150mM NaCl、10mM EDTA、0.1％NaN ₃ Ph=7.2), 50 μl of methotrexate derivative solution is added;

4. the above mixed solution was subjected to desalting treatment by shaking at room temperature (18-28deg.C, preferably 20-25deg.C) for 2-3 hours, and the resultant product was G6 PDH-methotrexate conjugate (concentration 0.1mM-2.0 mM).

2. Control coupling method A

G6 PDH-methotrexate conjugate was prepared according to the method disclosed in example four of CN 104569373A.

3. Control coupling method B (relying on activation of the self-contained group of methotrexate)

1. 2.38mL of glucose hexaphosphate dehydrogenase was dissolved in 12mL of Tris buffer, followed by sequential addition of 225mg of reduced nicotinamide adenine dinucleotide NADH,135mg of glucose-6-phosphate, and 2.25mL of dimethyl sulfoxide; the pH value of the Tris buffer solution is 9.0, and the concentration of each component is as follows: 0.05mol/L Tris, 3.3mM magnesium chloride, 145.4mM sodium chloride.

2. Activation of methotrexate derivatives: 10mg of methotrexate derivative is dissolved in 420. Mu.L of dimethyl sulfoxide and 180. Mu.L of dimethylformamide, 6. Mu.L of tributyl amine and 350. Mu.L of isobutyl chloroformate are added and stirred at 2-8 ℃ for 30 minutes;

3. mixing the solutions obtained in the steps 1 and 2, stirring for 12-16 hours at the temperature of 2-8 ℃, and purifying the coupled enzyme-labeled antigen by using a G-25 gel chromatographic column to obtain the glucose-6-phosphate dehydrogenase labeled methotrexate derivative.

EXAMPLE 3 preparation of the kit

The following kits for the detection of methotrexate were prepared, comprising:

1. preparation of the first reagent:

2. preparation of the second reagent:

3. calibration material: 20mM HEPES buffer, 0.2-0.8. Mu.M, 0.9-1.2. Mu.M, 1.3-2.0. Mu.M methotrexate (or added as needed);

4. quality control product: 20mM HEPES buffer, 0. Mu.M, 0.13. Mu.M, 0.25. Mu.M, 0.50. Mu.M, 1.00. Mu.M, 2.00. Mu.M methotrexate (or added as needed).

5. The above reagents (optionally including quality control substances and calibration materials) are assembled into a detection kit.

Test case

In a homogeneous reaction system, methotrexate and G6 PDH-methotrexate conjugate in a sample compete for binding to the anti-methotrexate antibody site simultaneously, and as the enzyme activity decreases after the antibody is bound with the conjugate, the more methotrexate in the sample is free, the more the antibody site competing for binding is, the less the antibody is bound with the enzyme conjugate, and the enzyme conjugate which is not bound with the antibody catalyzes the oxidation form (NAD ⁺ ) Converted into beta-nicotinamide adenine dinucleotide reduction (NADH), the concentration of the methotrexate in the sample is directly proportional to the generation amount of NADH, and the concentration of the methotrexate in the sample can be obtained through the change of absorbance.

TABLE 1 full automatic Biochemical instrument parameters

Test example 1. Performance of the kit of the present application

1. Scaling absorbance

Methotrexate calibrator was dissolved in buffer (0.9% NaCl,0.1% NaN) ₃ ) 6 concentrations of calibrator were prepared. The calibration working volume is 2-10 mu L, then 100-200 mu L of the first reagent and 50-100 mu L of the second reagent are added, the absorbance change rate of a specific reading point is measured by a velocity method, the dominant wavelength is 340nm, the auxiliary wavelength is 405nm, and a calibration curve is drawn. The establishment and optimization of the calibration curve in this application is done on Hitachi 7180, but other mainstream models (AU 680, attapulgite C16000, etc.) are available through detection.

2. Precision experiments

And (3) measuring high, medium and low quality control products and clinical samples by using the calibration curve established above.

TABLE 2 precision of

3. Linearity of

TABLE 3 linearities

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4. Airborne stability (D375C mutant)

Selecting quality control products with high, medium and low concentrations, detecting three times every day or every other day, and data show that the calibration period of the kit can be stabilized for more than two weeks; the reagents were placed on the machine after unsealing, the test showed on-board stability for more than 40 days, and the data are presented in table 6 (where 40 days represents the test data after 40 days of on-board recalibration).

5. Drug interference experiment

The 20 compounds and drugs commonly used are selected, and the methotrexate concentration is 10 mu M, and the compounds with the following concentrations have no obvious interference on the detection (D375C mutant) of the kit.

TABLE 4 drug interference experiment

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Detection example 2 inhibition of antibodies in conjugates

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-methotrexate conjugate, the activity of the G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing NAD to be converted into NADH is reduced, and the difference between an added antibody and an experimental group without the added antibody is compared by detecting the change of the NADH amount, wherein the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. Reaction system

TABLE 5 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-methotrexate conjugate when the antibody is added with the antibody is not added with the antibody, and obtaining the inhibition condition of the antibody on the G6 PDH.

Compared with the conjugate prepared by the published mutation site (A45C, K C), the enzyme mutant of the application has obviously improved antibody inhibition rate, and can reach more than 44.6% (G426C: 44.6%; D375C: 51%), up to 60.3% (D306C). Whereas the inhibition rate of the previously published mutation site (e.g. a45C, K55C) is 39.7% to 42.2%.

While not being limited to a particular theory, it may be explained in part as: in comparison with the G6PDH mutant (A45C, K C) in the prior art, the mutation site (i.e. the site for introducing free sulfhydryl) in the enzyme mutant is the coupling site with hapten (such as hormone, small molecule drug, etc.). When hapten is combined with hapten specific antibody at this position, the steric hindrance formed has a great influence on the activity of G6PDH enzyme, and after mutation is introduced, the steric folding of the molecule cannot be substantially influenced. Therefore, the position of this mutation site is very important, and it is necessary to combine the activity of the G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The mutant of the enzyme has obvious improvement on the inhibition rate of the antibody. After the conjugate of the enzyme mutant and methotrexate is prepared into a kit, the reagent has obvious performance improvement in the aspects of the inter-batch variation coefficient, linearity, specificity and the like.

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Claims (1)

1. Use of a conjugate in the preparation of a detection reagent, wherein:

the detection reagent is methotrexate;

the detection reagent is a homogeneous enzyme immunoassay detection reagent;

the conjugate is prepared from a glucose 6-phosphate dehydrogenase mutant and a methotrexate derivative according to a molar ratio of 1:1, covalent coupling;

the methotrexate derivative is represented by formula I:

wherein, the liquid crystal display device comprises a liquid crystal display device,

m is an integer from 1 to 10, preferably an integer from 1 to 5;

preferably, the methotrexate derivative is of formula II:

the glucose 6-phosphate dehydrogenase mutant comprises a D306C mutation compared to the wild-type glucose 6-phosphate dehydrogenase; the glucose 6-phosphate dehydrogenase mutant is shown in SEQ ID No. 2.

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