CN112285038A

CN112285038A - 6-phosphoglucose dehydrogenase mutant and application thereof in preparing digitoxin detection reagent

Info

Publication number: CN112285038A
Application number: CN201911372535.0A
Authority: CN
Inventors: 封建新; 张启飞; 龚俊; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2019-12-27
Publication date: 2021-01-29
Anticipated expiration: 2039-12-27
Also published as: CN117054643A; CN116718764A; CN116124721A; CN111650135A; CN111537451A; CN112285037A; CN116338215A; CN116840467A; CN111504920A; CN116718761A; CN116626281A; CN116735512A; CN116148198A; CN117030640A; CN111537452B; CN116359146A; CN116699122A; CN116773795A; CN111487207A; CN116008201A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a digoxigenin detection reagent. Specifically, the glucose-6-phosphate dehydrogenase mutant of the present application comprises one or a combination of mutations 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 and accurate quantification, and is suitable for high-throughput detection.

Description

6-phosphoglucose dehydrogenase mutant and application thereof in preparing digitoxin detection reagent

Priority of the present application claims patent application nos. 201910017764.4 filed on 2019, 1, 9 and 201910423122.4 mutant glucose-6-phosphate dehydrogenase and use thereof in the preparation of test agents filed on 2019, 5, 21, which are incorporated herein by reference.

Technical Field

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

Background

Haptens, some small molecular substances (molecular weight less than 4000Da), alone cannot induce an immune response, i.e. are not immunogenic, but can acquire immunogenicity when crosslinked or conjugated with carriers such as macromolecular proteins or non-antigenic polylysine, and induce an immune response. These small molecule substances can bind to response effector products, have antigenicity, are immunoreactive only and are not immunogenic, and are also called incomplete antigens.

The hapten can be combined with a corresponding antibody to generate an antigen-antibody reaction, and can not singly stimulate the human or animal body to generate the antigen of the antibody. It is immunoreactive only, has no immunogenicity, and is also called 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 will stimulate the production of corresponding antibodies in animals. Haptens, once bound to a protein, constitute an antigenic cluster of the protein. Some chemical active group substances (such as penicillin, sulfanilamide, etc.) with molecular weight smaller than that of general hapten and specific structure are called simple hapten.

Small molecule antigens or haptens lack two or more sites that can be used in sandwich assays, and therefore cannot be measured using the double antibody sandwich assay, and often use a competition mode. The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete to bind with the solid-phase antibody. The more the amount of the antigen in the specimen is, the less the enzyme-labeled antigen bound to the solid phase is, and the lighter the color develops. ELISA measurement of small molecule hormone, medicine, etc. is used in different methods.

Digitoxin (DG) has the following structural formula:

digitoxin belongs to a class of naturally occurring Cardiac Glycosides (CG) obtained from digitalis, digitalis or other cardiac glycosides suitable for being obtained from digitalis.

A large amount of digitoxin is rapidly and completely absorbed in the gastrointestinal tract, the metabolism in the body is slow, the digitoxin is metabolized in the liver, and most metabolites are inactive. Digoxin is commonly used in heart failure, and indications are congestive heart failure and arrhythmia, which are long-lasting due to its slow action and are particularly suitable for long-term administration to patients with chronic cardiac insufficiency. Digitalis glycosides and Na⁺/K⁺Reversible binding of ATPase to the cell membrane, prevention of binding of the enzyme to ATP, inhibition of Na⁺And K⁺Active transport of (3) to make Na in the cell⁺Increase, K⁺Reduction, which is due to the direct electrophysiological effects and toxicity of digitosides.

The dosage of digitoxin should be carefully adjusted according to individual needs of patients. Digitoxin therapeutic steady state plasma concentrations range from 10 to 25ng/mL, with higher concentrations (30ng/mL) possibly being associated with toxicity. Therefore, there is a clinical need for effective monitoring of digoxigenin concentrations in patients.

The currently known digitoxin detection methods mainly comprise: chemiluminescence immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, these detection methods have many defects, for example, although chemiluminescence has good sensitivity, a special device is required, and the cost for use is high, which is not favorable for popularization. In the clinical detection and diagnosis process, homogeneous enzyme immunoassay (EMIT) and latex enhanced immunoturbidimetry are mainly used for detection.

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, an enzyme-labeled antigen (such as G6 PDH-digoxigenin) and a non-labeled antigen (digoxigenin) compete with and are combined with a quantitative antibody (digoxigenin antibody), when the antibody is more combined with the non-labeled antigen, the more activity released by the enzyme-labeled antigen is, the more NADH is generated by catalyzing a substrate NAD +, and the change of absorbance of NADH is detected under the wavelength of 340nm, so that the content of the digoxigenin in the liquid can be calculated.

The existing homogeneous enzyme immunoassay method and latex agglutination turbidimetry method are limited in application due to complex preparation process and large batch difference. CN102768284A describes a preparation method of a conjugate of small molecule drug G6PDH enzyme. However, the prior art methods rely on the activation of reactive groups carried by the small molecule drug itself, followed by reaction with an enzyme. Such a strategy is difficult to ensure a directed reaction between small molecule drugs and enzymes, resulting in large batch-to-batch variation.

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 digoxigenin detection kit.

According to some embodiments, a glucose-6-phosphate dehydrogenase mutant is provided. In contrast to the previously published mutant of glucose-6-phosphate dehydrogenase of patent US006090567A (halogenated immunological systems using mutant glucose-6-phosphate dehydrogenes), the glucose-6-phosphate dehydrogenase mutant of the present application comprises a mutation selected from the group consisting of: D306C, G426C, D375C.

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, there is provided a polynucleotide encoding a glucose-6-phosphate dehydrogenase mutant of the present application.

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

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

In some embodiments, n is 1 to 50, e.g., 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.

In some specific embodiments, the glucose-6-phosphate dehydrogenase mutant of the present application is preferably present in a molar ratio of 1: 1.

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

According to the present application, the skilled person will understand that "hapten" also comprises forms of its derivatives. To facilitate conjugation to glucose-6-phosphate dehydrogenase, haptens (e.g., digoxigenin) that do not themselves bear a coupling group (e.g., a group that reacts with a thiol group) can be engineered to bear a linker for covalent binding to the thiol group. Thus, in the present application, a hapten derivative refers to a hapten which has been engineered to carry a thiol-reactive group.

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

Haptens are exemplified by, but not limited to: theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxigenin), mycophenolic acid, rapamycin, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, cotinine, uromorphine, derivatives of urotensin, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental prolactin, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, cortisol, urinary 17-hydroxysteroids, corticosteroids, cardiac glycosides, morphine, vitamin A, vitamin B, Urinary 17-ketosteroids, dehydroepiandrosterone and sulfate esters, aldosterone, urovanillyl mandelic acid, 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-one prostaglandin F1 alpha, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, sodium, adenosine cyclophosphate, guanosine cyclophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

In a particular embodiment, the hapten is digoxigenin or a derivative thereof.

In particular embodiments, the hapten is a digoxigenin derivative bearing a thiol-reactive group, such as, for example, a maleimide, bromoacetyl, vinyl sulfone, or aziridine.

In a particular embodiment, the hapten is a digoxigenin derivative, represented by formula I:

wherein ,

in some embodiments, m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6, such as 1, 2, 3,4, 5, 6.

In some embodiments, X is maleimide, bromoacetyl, vinyl sulfone, or aziridine.

The skilled artisan will appreciate that X functions to react with the sulfhydryl group of glucose-6-phosphate. Covalent bonding of maleimide, bromoacetyl, vinyl sulfone, aziridine, and thiol groups is contemplated. Although specific groups are employed in the examples, they are not intended to be limiting.

In some specific embodiments, the digitoxin derivative has a structure selected from the group consisting of:

m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6.

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

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

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

In specific embodiments, the detection reagent is selected from the group consisting of: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

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

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

-a first reagent comprising a substrate, a buffer and a digoxigenin 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, 0ng/ml to 80ng/ml digoxigenin; and

-optionally, a quality control comprising 10mM to 500mM buffer, 10ng/ml to 50ng/ml digoxigenin.

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

10ng/ml to 10 mu g/ml digitoxin antibody,

0.1 to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer solution,

0.01. mu.g/ml to 10. mu.g/ml of a conjugate according to the application,

0.1 to 5g/L stabilizer,

0.1g/L to 5g/L of surfactant,

0.1g/L to 5g/L preservative.

In some embodiments, the buffer is selected from one or a combination of: tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution; preferably, a phosphate buffer; the concentration of the buffer solution is 10mmol/L to 500mmol/L, preferably 100 mM; the pH of the buffer is 7 to 8.

In some embodiments, the stabilizing agent is selected from one or a combination of: bovine serum albumin, trehalose, glycerol, sucrose, mannitol, glycine, arginine, polyethylene glycol 6000, polyethylene glycol 8000; bovine serum albumin is preferred.

In some embodiments, the surfactant is selected from one or a combination of: brij35, Triton X-100, Triton X-405, Tween20, Tween30, Tween80, coconut oil fatty acid diethanolamide, AEO7, preferably Tween 20.

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

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

In some specific embodiments, the digoxigenin antibody is derived from: mouse, rat, cat, dog, primate, cow, horse, sheep, camelid, avian, human.

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

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

1) providing a digoxigenin derivative according to the present application, in particular in an aprotic solvent (such as 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, buffer pH between 6.0 and 8.0);

3) (ii) mixing the glucose-6-phosphate dehydrogenase mutant and the digoxigenin derivative at a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow the digoxigenin derivative and the glucose-6-phosphate dehydrogenase mutant to couple, resulting in the seed conjugate;

4) the conjugate is optionally subjected to purification, such as desalting treatment or the like, as required.

In some specific embodiments, steps 1) and 2) are interchangeable.

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

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

Drawings

FIG. 1 shows the structure of digitoxin.

FIG. 2 shows the structure of digoxigenin derivatives.

FIG. 3A. G6PDH (wild-type) amino acid sequence (SEQ ID No. 1); derived from Leuconostoc pseudomesenteroides of Leuconostoc.

FIG. 3B. G6PDH (D306C) amino acid sequence (SEQ ID No. 2).

FIG. 3C.G6PDH (D375C) amino acid sequence (SEQ ID No. 3).

FIG. 3D.G6PDH (G426C) amino acid sequence (SEQ ID No. 4).

Detailed Description

Examples

Example 1 Synthesis of Didigoxin derivatives

1. Synthesis of Compound 2

1.0g of digoxigenin was dissolved in 80ml of 95% ethanol, and then 10ml of an aqueous periodic acid solution (1.0g of periodic acid) was added thereto, followed by stirring at room temperature (18 to 28 ℃, preferably 20 to 25 ℃) for 1 hour.

The residue was removed by filtration, the solvent was removed under reduced pressure, and the mixture was extracted with dichloromethane. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to give compound 2 (white solid, 0.90g, yield 90%).

2. Synthesis of Compound 4

Compound 2(900mg, 1.18mmol) was dissolved in 10ml of dry methanol, and compound 3(318mg, 1.0mmol) was added to the reaction system, followed by stirring at room temperature for 5 minutes. Sodium cyanoborohydride (146mg, 2.32mmol) was added and stirred at room temperature for about 12 hours.

The solvent was removed under reduced pressure and purified by direct column chromatography to give compound 4 (white solid, 400mg, 46%).

3. Synthesis of Compound 5

Compound 4 was dissolved in 15ml of dichloromethane, and stirred at room temperature for 30 minutes under nitrogen, then 10ml of piperidine was added, and stirred at room temperature for 2 hours. The solution was removed under reduced pressure and purified by column chromatography to give compound 5(500mg, 85%).

4. Synthesis of digitoxin derivatives

Compound 5(88mg, 0.11mmol) and compound 6(17mg, 0.11mmol) were dissolved in 8mL DCM, to which triethylamine (33mg, 0.33mmol) was added dropwise, followed by HATU (50mg, 0.13mmol) and stirred at room temperature for 2h to give the digoxigenin derivative (white solid, 48mg, 43%).

The structure of the product was confirmed by a conventional method. This example allows digoxigenin to carry a group that can bind to an enzyme.

Example 2 coupling of Didigoxin derivatives to G6PDH molecules

The G6 PDH-digoxigenin conjugate according to the present application was coupled as follows: a thiol-reactive group (such as, but not limited to, a maleimide group) on the digoxigenin derivative molecule is covalently bound to a thiol on the G6PDH molecule.

1. Solution preparation:

digitoxin derivative solution: digoxigenin derivative prepared in example 1, 10mg/ml, was dissolved in DMF;

g6PDH solution: g6PDH (mutant of the present application or prior art mutant) was dissolved in PB 100mmol, NaCl 100mmol, pH 8.0;

coupling solution: 100mM PB/K, 100mM EDTA, 150mM NaCl, pH 7.2;

desalting solution: 100mM PB/K, 0.1% NaN₃、1％NaCl，pH＝8.0。

2. Coupling operation:

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

3. And (3) oscillating the reaction system at room temperature for 4h, eluting with the desalting solution by using a desalting column, and collecting a protein peak to obtain a product, namely the G6 PDH-digitoxin conjugate.

Example 3 preparation of the kit

The following kit for detecting digoxigenin is prepared, which comprises:

reagent R1, comprising:

50mM HEPES，pH 7.0

10mM glucose-6-phosphate

10mM beta-nicotinamide adenine dinucleotide

50ng/ml digoxigenin antibody (commercially available antibody, without particular limitation)

1g/L bovine serum albumin

1g/L Tween20

1g/L sodium azide;

reagent R2, comprising:

200mM Tris buffer, pH 8.0

0.1. mu.g/ml G6 PDH-digitoxin conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

calibration products: 20mM HEPES buffer, and 0.0, 5.0, 10.0, 20.0, 40.0, 80.0ng/ml digitoxin (or added as needed);

quality control product: 20mM HEPES buffer, and 8ng/ml, 15ng/ml, 35ng/ml digitoxin (or added as needed).

And assembling the reagents (optionally containing quality control products and calibration products) into the digoxigenin homogeneous enzyme immunoassay kit.

Example of detection

TABLE 1 parameters of fully automatic biochemical analyzer

Model type	Hitachi 7180
		Analysis point	[Rate-A][10][25][34]
Wavelength (SUB/MAIN)	[410]/[340]
		S.VIL	[10]
S.R1；S.R3	[150]；[50]
		ABS.LIMIT	[32000][ increasing by one]
Calibration type	[Spline]
		POINT	[6]SPAN PONIT[6]
Calibration article	0.0、5.0、10.0、20.0、40.0、80.0ng/ml
		Sample(s)	The sample to be tested is various physiological samples, such as serum and plasma

Test example 1 accuracy, precision, Linear assay of the kit of the present application (D306C mutant)

TABLE 2 accuracy, precision

TABLE 3 linearity

	Side 1	Side 2	Side 3	Mean value	Theoretical value	Relative deviation of	Absolute deviation
								1	1.81	2.08	1.68	1.86	1.28	-	0.58
2	7.30	7.19	7.66	7.38	7.38	0.1％	0.01
								3	13.22	12.90	12.83	12.98	13.48	-3.7％	-0.49
4	20.16	20.45	19.59	20.07	19.58	2.5％	0.49
								5	24.39	25.65	24.89	24.98	25.68	-2.7％	-0.70
6	32.00	30.43	31.24	31.22	31.78	-1.7％	-0.55
								7	38.84	39.04	39.20	39.03	37.87	3.0％	1.15
8	44.62	42.71	44.01	43.78	43.97	-0.4％	-0.19
								9	48.80	51.18	50.15	50.04	50.07	-0.1％	-0.03
10	57.52	54.13	54.70	55.45	56.17	-1.3％	-0.72
								11	62.24	61.35	61.54	61.71	62.27	-0.9％	-0.56
12	67.93	68.98	69.58	68.83	68.37	0.7％	0.46
								13	74.25	78.44	72.43	75.04	74.47	0.8％	0.57

Detection example 2 recovery experiment

The use of United States Pharmacopoeia (USP) digitoxin pure product added to the serum mixture samples, preparation of 4 concentrations, each level 21 times. And calculating the mean value and the deviation.

TABLE 4 recovery experiment

Detection example 3 anti-interference of common drugs

TABLE 5 anti-interference measurement results

Numbering	Interfering substance	Concentration (μ g/ml)
			1	N-acetylcysteine	150
2	Amitriptyline	2
			3	Ampicillin sodium	100
4	K-hydroxybenzenesulfonate (Hydroquinone sulfonic acid potassium salt)	200
			5	Hydroxyzine dihydrochloride	1
6	Methyldopa sesquihydrate	20
			7	Promethazine	100
8	Ascorbic acid	30
			9	Tetracycline derivatives	50
10	Acetylsalicylic acid	1000
			11	Probenecid's sodium salt	500
12	Levodopa (3, 4-dihydroxy-L-phenylalanine)	20
			13	Metronidazole	100
14	Phenothiazines	200
			15	Ibuprofen	500
16	Baotasone	16
			17	Acetaminophen	200
18	Chlorpromazine	100
			19	Artesunate (Artesunate)) Merr.)	50
20	Nortriptyline	1
			21	Cefoxitin	2500
22	Cetirizine dihydrochloride	3
			23	Cyclosporin	5
24	Desipramine	3
			25	Ethylbenzene production	50
26	5- (P-hydroxyphenyl-5-phenylhydantoins)	1000
			27	Imipramine	6
28	Phenobarbital	500
			29	Phenytoin sodium	500
30	Theophylline	100
			31	Valproic acid	1000
32	Ethosuximide	1000
			33	"Xiadengtong	1000
34	Rifampicin	60

Detection example 4 Cross reaction

And (3) dissolving the digitoxin pure product by using a buffer solution to prepare digitoxin calibration products with different concentrations. Digoxigenin analogs were added to the pooled serum samples at the following concentrations, respectively.

The reagent uses digoxigenin calibrator calibration to measure the mixed serum sample and the sample for 5 times respectively, and the ratio of the measured value of the sample to the measured value of the mixed serum sample is the cross reaction rate.

TABLE 6 Cross-reactivity

Chemical name	Reagent for the present application
		5.00ng/ml digitoxin aglycone	309.7％
20ng/mL digitoxin	2.43％
		20ng/mL Didigoxin glycoside (digoxigenin-Mono)	0.08％
20ng/mL Didigitoxin glycoside (digoxigenin-bis)	1.60％
		20ng/mL digoxin	1.05％
750ug/mL Convolvulus glycoside	1.85％
		750ug/mL deacetyl hairy flower glycoside	0.33％

Detection example 5 detection kit for Digitalis glycosides

Three batches of the reagent of the present application (containing the D306C mutant) were used for separate calibration to calculate the difference in absorbance change between the different batches.

TABLE 7 calibration data between batches

TABLE 8 comparison between batches

The control kit (which differs from the present kit in that the enzyme is replaced with the prior art a45C mutant) was tested in the same way for run-to-run differences with CV in the range of 2.9% to 4.5%, significantly lower than the present kit.

Test example 6 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-digoxigenin conjugate, the activity of G6PDH enzyme is influenced due to steric hindrance, so that the efficiency of catalyzing conversion from NAD to NADH is reduced, and the difference between an experimental group with the antibody added and an experimental group without the antibody added is compared by detecting the change of NADH amount, and the difference is reflected in the inhibition capacity of the antibody on G6 PDH.

2. Reaction system:

TABLE 9 preparation of reagents for detection of antibody inhibition

3. As a result:

and comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance value of the G6 PDH-digitoxin conjugate to obtain the inhibition condition of the antibody on G6 PDH.

The antibody inhibition rate was ═ 1- (change in absorbance of G6 PDH-digoxigenin with antibody/change in absorbance of G6 PDH-digoxigenin without antibody) ] × 100%.

Compared with the published mutation site (A45C), the mutant of the application has obviously improved antibody inhibition rate which can reach more than 32 percent (G426C: 32%; D375C: 47%) and can reach as high as 55 percent (D306C). Whereas the inhibition rates of previously published mutation sites (e.g. a45C, K55C) ranged from 30 to 44%.

While not being bound to a particular theory, it may be partially explained as:

compared with the G6PDH mutant (A45C, K55C) in the prior art, the mutant (especially D306C, D375C) of the enzyme of the application is a site where the mutation site (i.e. the site for introducing free thiol) is coupled with hapten (such as hormone, small molecule drug and the like). When the hapten binds to a hapten-specific antibody at this position, the steric hindrance formed has minimal effect on the activity of the G6PDH enzyme, and after the introduction of the mutation, it does not substantially affect the steric folding of the molecule. Therefore, the position of this mutation site is very important, and needs to be compatible with the activity of G6PDH enzyme, the spatial folding of the coupling molecule, and the sufficient exposure of the hapten epitope.

The enzyme mutant has obviously improved antibody inhibition rate. After the conjugate obtained by coupling the enzyme mutant and the digitoxin is prepared into the kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, specificity and the like.

Sequence listing

<110> Beijing Jiuqiang Biotechnology Ltd

<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of digoxigenin detection reagent

<130> 390265CG

<150> 201910017764.4

<151> 2019-01-09

<150> 201910423122.4

<151> 2019-05-21

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 486

<212> PRT

<213> Leuconostoc pseudomesenteroides (Leuconostoc pseudosensoides)

<400> 1

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 2

<211> 486

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (306)..(306)

<223> G6PDH mutant, D at position 306 was replaced with C compared to wild type

<400> 2

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Cys Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 3

<211> 486

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (375)..(375)

<223> G6PDH mutant, D at position 375 was replaced with C compared to wild type

<400> 3

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Cys Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

<210> 4

<211> 486

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<222> (426)..(426)

<223> G6PDH mutant, G at position 426 was replaced with C compared to wild type

<400> 5

Met Val Ser Glu Ile Lys Thr Leu Val Thr Phe Phe Gly Gly Thr Gly

1 5 10 15

Asp Leu Ala Lys Arg Lys Leu Tyr Pro Ser Val Phe Asn Leu Tyr Lys

20 25 30

Lys Gly Tyr Leu Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln

35 40 45

Ala Leu Asn Asp Asp Glu Phe Lys Gln Leu Val Arg Asp Ser Ile Lys

50 55 60

Asp Phe Thr Asp Asp Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe

65 70 75 80

Ser Tyr Arg Ala His Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Leu

85 90 95

Lys Glu Ala Ile Glu Glu Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn

100 105 110

Arg Ile Phe Tyr Met Ser Val Ala Pro Arg Phe Phe Gly Thr Ile Ala

115 120 125

Lys Tyr Leu Lys Ser Glu Gly Leu Leu Ala Asp Thr Gly Tyr Asn Arg

130 135 140

Leu Met Ile Glu Lys Pro Phe Gly Thr Ser Tyr Asp Thr Ala Ala Glu

145 150 155 160

Leu Gln Asn Asp Leu Glu Asn Ala Phe Asp Asp Asn Gln Leu Phe Arg

165 170 175

Ile Asp His Tyr Leu Gly Lys Glu Met Val Gln Asn Ile Ala Ala Leu

180 185 190

Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr Ile

195 200 205

Lys Asn Val Gln Val Thr Leu Ser Glu Val Leu Gly Val Glu Glu Arg

210 215 220

Ala Gly Tyr Tyr Asp Thr Ala Gly Ala Leu Leu Asp Met Ile Gln Asn

225 230 235 240

His Thr Met Gln Ile Val Gly Trp Leu Ala Met Glu Lys Pro Glu Ser

245 250 255

Phe Thr Asp Lys Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala

260 265 270

Leu Lys Ile Tyr Asp Glu Ala Glu Val Asn Lys Tyr Phe Gly Arg Ala

275 280 285

Gln Tyr Gly Ala Gly Asp Ser Ala Asp Phe Lys Pro Tyr Leu Glu Glu

290 295 300

Leu Asp Val Pro Ala Asp Ser Lys Asn Asn Thr Phe Ile Ala Gly Glu

305 310 315 320

Leu Gln Phe Asp Leu Pro Arg Trp Glu Gly Val Pro Phe Tyr Val Arg

325 330 335

Ser Gly Lys Arg Leu Ala Ala Lys Gln Thr Arg Val Asp Ile Val Phe

340 345 350

Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu Gln Glu Ala Gln Glu Ala

355 360 365

Val Leu Ser Ile Ile Ile Asp Pro Lys Gly Ala Ile Glu Leu Lys Leu

370 375 380

Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr Ile Asp Leu

385 390 395 400

Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu Pro Tyr

405 410 415

Glu Arg Met Ile His Asp Thr Met Asn Cys Asp Gly Ser Asn Phe Ala

420 425 430

Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala Ile Ser

435 440 445

Ala Val Tyr Thr Ala Asp Lys Ala Pro Leu Glu Thr Tyr Lys Ser Gly

450 455 460

Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly Asp

465 470 475 480

Ala Trp Val Phe Lys Gly

485

Claims

1. A conjugate which is formed by coupling a 6-phosphoglucose dehydrogenase mutant and a hapten;

the hapten is digoxigenin or a derivative thereof;

preferably, the digitoxin derivative is represented by formula I:

wherein ,

m is an integer from 0 to 20, preferably an integer from 1 to 10, preferably an integer from 1 to 6;

x is selected from: maleimide, bromoacetyl, vinyl sulfone, aziridine;

more preferably, X is maleimide.

2. The conjugate of claim 1, wherein:

the glucose-6-phosphate dehydrogenase mutant comprises a mutation selected from the group consisting of: D306C, D375C, G426C;

preferably, the glucose-6-phosphate dehydrogenase mutant is represented by a sequence selected from the group consisting of: SEQ ID No.2, SEQ ID No.3, SEQ ID No. 4.

3. The conjugate of claim 1 or 2, wherein the digoxigenin derivative has a structure selected from the group consisting of:

4. A reagent comprising the conjugate of any one of claims 1 to 3.

5. Use of a conjugate according to any one of claims 1 to 3 in the preparation of a detection reagent:

the detection reagent is a digitoxin detection reagent;

preferably, the detection reagent is selected from: enzyme-linked immunosorbent assay reagent, chemiluminescence immunoassay reagent, homogeneous enzyme immunoassay reagent and latex enhanced immunoturbidimetry reagent.

6. A digitoxin assay kit, comprising:

a first reagent comprising a substrate, a digoxigenin antibody, a buffer;

a second reagent comprising the conjugate of any one of claims 1 to 3, a buffer;

optionally, a calibrator comprising 10mM to 500mM buffer, 0ng/ml to 80ng/ml digitoxin; and

optionally, a quality control comprising 10mM to 500mM buffer, 10ng/ml to 50ng/ml digoxigenin.

7. The digitoxin assay kit of claim 6, comprising:

a first reagent comprising:

10mM to 500mM, preferably 100mM to 300mM, buffer,

5mM to 50mM, preferably 10mM to 20mM, glucose-6-phosphate,

5mM to 50mM, preferably 10mM to 20mM, oxidized form beta-nicotinamide adenine dinucleotide,

10ng/ml to 10. mu.g/ml, preferably 20ng/ml to 200ng/ml, of digoxigenin antibodies,

0.1 to 5g/L, preferably 1 to 5g/L, of a stabilizer,

0.1g/L to 5g/L, preferably 1g/L to 5g/L, of a surfactant,

0.1g/L to 5g/L, preferably 1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM, preferably 100mM to 300mM, buffer,

The conjugate according to any one of claims 1 to 3, comprising 0.01 to 10 μ g/ml, preferably 0.05 to 0.5 μ g/ml,

0.1 to 5g/L, preferably 1 to 5g/L, of a stabilizer,

0.1g/L to 5g/L, preferably 1g/L to 5g/L, of a surfactant,

0.1g/L to 5g/L, preferably 1g/L to 5g/L preservative;

the buffer is selected from one or a combination of the following: tromethamine buffer solution, phosphate buffer solution, Tris-HCl buffer solution, citric acid-sodium citrate buffer solution, barbital buffer solution, glycine buffer solution, borate buffer solution and trimethylolmethane buffer solution;

preferably, the buffer of the first reagent is HEPES buffer;

preferably, the buffer of the second reagent is Tris buffer;

the pH of the buffer is 7 to 8;

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;

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 Tween 20;

the preservative is selected from one or a combination of the following: azide, MIT, PC-300, thimerosal;

the azide is selected from: sodium azide and lithium azide.

8. The digitoxin assay kit of claim 6, comprising:

a first reagent comprising:

50mM HEPES，pH 7.0、

10mM glucose-6-phosphate,

10mM oxidized beta-nicotinamide adenine dinucleotide,

50ng/ml digitoxin antibody,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide;

a second reagent comprising:

200mM Tris buffer, pH 8.0,

0.1. mu.g/ml of the conjugate according to any one of claims 1 to 3,

1g/L bovine serum albumin,

1g/L Tween20、

1g/L sodium azide.

9. A method of preparing a conjugate comprising the steps of:

providing a glucose-6-phosphate dehydrogenase mutant as defined in any one of claims 1 to 3;

providing digitoxin or a derivative thereof;

the glucose-6-phosphate dehydrogenase mutant is coupled with the digoxigenin or the derivative thereof;

preferably, the digitoxin derivative is represented by formula I:

wherein ,

x is selected from: maleimide, bromoacetyl, vinyl sulfone, aziridine;

more preferably, X is maleimide.

10. A method of preparing a conjugate according to claim 9, comprising the steps of:

1) providing a digoxigenin derivative, preferably in an aprotic solvent;

2) providing a glucose-6-phosphate dehydrogenase mutant as defined in any one of claims 1 to 3, preferably in a buffer;

3) contacting the glucose-6-phosphate dehydrogenase mutant and the digoxigenin derivative at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that the digoxigenin derivative and the glucose-6-phosphate dehydrogenase mutant are conjugated to give the conjugate;

4) optionally, purifying, preferably desalting, the conjugate;

steps 1) and 2) are interchangeable;

the buffer is selected from: PBS, Tris, TAPS, TAPSO, pH of the buffer is 6.0 to 8.0;

the aprotic solvent is selected from one or a combination of the following: acetonitrile, dimethylformamide, dimethyl sulfoxide;

preferably, prior to step 3), the glucose-6-phosphate dehydrogenase mutant comprises one or more free sulfhydryl groups; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375 or 426.