CN111504920A

CN111504920A - 6-glucose phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent

Info

Publication number: CN111504920A
Application number: CN201911404154.6A
Authority: CN
Inventors: 封建新; 张启飞; 龚俊; 王贵利; 刘希
Original assignee: Beijing Strong Biotechnologies Inc
Current assignee: Beijing Strong Biotechnologies Inc
Priority date: 2019-01-09
Filing date: 2019-12-31
Publication date: 2020-08-07
Anticipated expiration: 2039-12-31
Also published as: CN111650135A; CN111693473A; CN116626281A; CN116735512A; CN116297271A; CN111239060B; CN112285037A; CN116840467A; CN116359146A; CN111487208A; CN116718761A; CN115791649A; CN111504920B; CN116840462A; CN110174363A; CN116699125A; CN115808398A; CN111678874A; CN111504921A; CN116338215A

Abstract

The application relates to a 6-phosphoglucose dehydrogenase mutant and application thereof in preparing a cortisol 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-glucose phosphate dehydrogenase mutant and application thereof in preparation of cortisol detection reagent

The present application claims priority from 201910017764.4 filed on day 1/9 in 2019 and 201910423122.4 "glucose-6-phosphate dehydrogenase mutant and its use in the preparation of test agents" filed on day 21/5 in 2019, 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 cortisol 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.

The principle is that the antigen in the specimen and a certain amount of enzyme-labeled antigen compete for binding with solid-phase antibody, the more the content of the antigen in the specimen is, the less the enzyme-labeled antigen is bound on the solid phase, the lighter the color is, and the more the E L ISA is used for measuring small molecular hormone, medicine, etc.

Cortisol (Cortisol) has the following structural formula:

cortisol, also called hydrocortisone or compound f (compound f), is an adrenocortical hormone (one of glucocorticoids) extracted from the adrenal cortex and having the strongest effect on carbohydrate metabolism.

Cortisol is produced from 11-deoxycorticosterol by the action of 11 β -hydroxylase in adrenal cortico mitochondria Cortisol can also be converted to cortin by the action of 11- β -hydroxysteroid dehydrogenase (11- β -hydroxysteroid dehydrogenase).

Cortisol plays an important role in the regulation of many important physiological processes, including energy metabolism, maintenance of electrolyte and blood pressure balance, immune regulation, stress response, cell proliferation and differentiation, memory regulation, and cognitive functions, among others. Cortisol in the blood binds mainly to corticosteroid-binding globulin and albumin, free cortisol only accounts for 3-5%, and cortisol concentration levels vary periodically every day, reaching a minimum in the first half of the night and a peak in the early morning.

The measurement of cortisol in blood is mainly used for human diseases such as hypersecretion of cortisol caused by cushing's syndrome and cortisol deficiency caused by addison's disease, and for treatment monitoring (dexamethasone inhibitory therapy and hormone replacement therapy).

Currently known cortisol detection methods are mainly: enzyme-linked immunosorbent assay, 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-cortisol) and a non-labeled antigen (cortisol) compete for binding with a quantitative antibody (cortisol antibody), when the antibody is more bound with the non-labeled antigen, the more activity is released by the enzyme-labeled antigen, the more NADH is generated by catalyzing a substrate NAD +, and the change of absorbance of NADH is detected at a wavelength of 340nm, so that the content of the cortisol 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.

The prior art describes a cortisol derivative-G6 PDH conjugate and a preparation method thereof:

1) dissolving G6PDH in Tris and MgCl solution at room temperature₂And NaCl (pH 9.0);

2) adding NADH, glucose 6 phosphate and carbitol;

3) then adding dimethyl sulfoxide dropwise;

4) weighing cortisol derivative in anhydrous state, and dissolving in DMF; reducing the temperature of the solution to-2 to-8 ℃; adding tributylamine;

5) adding isobutyl chloroformate, and stirring at-2 to-8 ℃ for 30 minutes;

6) dropwise adding the activated cortisol derivative solution into the dissolved G6PDH solution, and stirring at 2-8 ℃ overnight;

7) purifying the solution obtained in step 6) by a chromatographic column to obtain a final product of a conjugate of a G6 PDH-cortisol derivative (for example, but not limited to, the method described in CN 105131105A).

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 makes it difficult to ensure orientation between small molecule drugs and enzymes 1: 1, resulting in large batch-to-batch variation.

Disclosure of Invention

In view of the need in the art, the present application provides a novel mutant of glucose-6-phosphate dehydrogenase and its use in the preparation of a cortisol assay 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 coupling to glucose-6-phosphate dehydrogenase, haptens (e.g., cortisol) 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 include, but are not limited to, theophylline, phenytoin, vitamin D, 25 hydroxyvitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycosides (including digoxigenin), mycophenolic acid, rapamine, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, cotinine, urocortin, derivatives of urotensin, neuropeptide tyrosine, plasma galangin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactogen, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuresis hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, free thyroxine, free triiodothyronine, cortisone, cortisol, urinary 17-hydroxycorticosterol, urinary 17-ketosteroid, dehydroepiandrosterone and sulfatide, testosterone, angioneurotrophin, renin-D, renin-5, renin-angiotensin A, renin-5, renin-angiotensin, renin-5, renin-angiotensin, renin-5, renin-angiotensin, renin-5, renin-angiotensin, renin-angiotensin.

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

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

In a particular embodiment, the hapten is a cortisol derivative, as shown in formula I:

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 cortisol 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 a glucose-6-phosphate dehydrogenase mutant of the present application in the preparation of a cortisol detection reagent.

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

In particular embodiments, the detection device may be prepared in the form of a well plate (e.g., a 96-well plate), such as a plate coated with a reagent 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 cortisol detection kit comprising:

-a first reagent comprising a substrate, a buffer and a cortisol 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 800ng/ml cortisol; and

-optionally, a quality control comprising 10mM to 500mM buffer, 100ng/ml to 500ng/ml cortisol.

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

a first reagent comprising:

10mM to 500mM buffer solution,

5mM to 50mM substrate,

10ng/ml to 10 mu g/ml of cortisol antibody,

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

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

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

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

0.1 g/L to 5 g/L of preservative.

In some embodiments, the buffer is selected from one or a combination of tromethamine buffer, phosphate buffer, Tris-HCl buffer, citric acid-sodium citrate buffer, barbiturate buffer, glycine buffer, borate buffer, trimethylolmethane buffer, preferably phosphate buffer, at a concentration of 10 mmol/L to 500 mmol/L, preferably 100mM, and at a pH of 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, biological preservative PC (e.g. PC-300), thimerosal; the azide is selected from: sodium azide and lithium azide.

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

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

In some specific embodiments, the cortisol 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 cortisol 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) mixing the glucose-6-phosphate dehydrogenase mutant and the cortisol derivative at a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow the cortisol derivative and the glucose-6-phosphate dehydrogenase mutant to conjugate to give the seed conjugate;

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

In some embodiments, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein 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 other embodiments, the contacting molar ratio of the enzyme to the hapten in the reaction system is 1: n, wherein n is 0.01 to 1, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9.

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

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 cortisol.

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 cortisol.

FIG. 2 shows the structure of cortisol derivatives.

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

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).

Fig. 4. decap stability.

Detailed Description

Examples

Example 1 Synthesis of Cortisol derivatives

Cortisol (200mg, 0.55mmol) and Compound 2(78mg, 2.20mmol) were dissolved in 10ml of methanol with exclusion of light, and reacted for 5 minutes with exclusion of light.

The reaction system was heated to 50 ℃ and Compound 2(78mg, 2.20mmol) and Compound 3(60mg, 0.55mmol) were added to the reaction system, and the reaction was allowed to proceed for 5 minutes with incubation. The reaction system becomes colorless or pale yellow basically, the solvent is removed under reduced pressure, and the product is purified by column chromatography to obtain 180mg of product with 75 percent of yield.

Compound 5(100mg, 0.23mmol) and compound 6(53mg, 0.23mmol) were dissolved in DCM (5m L), triethylamine (70mg, 0.69mmol) was added dropwise thereto, HATU (105mg, 0.28mmol) was added, and the mixture was stirred at room temperature (18 to 28 ℃ C., preferably 20 to 25 ℃ C.) for 5h, and the solvent was removed under reduced pressure to give a cortisol derivative (80mg, 57%).

The structure of the product was confirmed by a conventional method.

This example allows cortisol to have a group that can bind to the enzyme.

Example 2 coupling of Cortisol derivatives to G6PDH molecules

First, the testing method of the present application

The coupling was carried out according to the G6 PDH-cortisol conjugate of the present application in the following manner: thiol-reactive groups (such as but not limited to maleimide groups) on the cortisol derivative molecule covalently bind to thiols on the G6PDH molecule.

1. Solution preparation:

cortisol derivative solution: 10mg/ml of the cortisol derivative prepared in example 1 is dissolved in DMF;

g6PDH solution: g6PDH (mutant of the present application or prior art mutant) was dissolved in PB 100mmol, NaCl100mmol, 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 cortisol 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-cortisol conjugate.

Second, control coupling method (refer to CN105131105A method to make the experimental protocol)

1. 15mg of 100KU G6PDH was dissolved in 12M L containing 72.6mg (0.05M) Tris and 8mgMgCl at room temperature₂(3.3mM) and 100mg NaCl (pH 9.0);

2. 225mg of NADH, nicotinamide adenine dinucleotide in reduced form, 135mg of glucose-6-phosphate and 0.75m of L carbitol were added to the beaker;

3. 2m L dimethylsulfoxide was added dropwise into the beaker;

4. weighing 10mg cortisol derivative in anhydrous state, dissolving in 600 μ L DMF, cooling to-2 to-8 deg.C, adding 3 μ L tributylamine;

5. adding 1.5 mu L isobutyl chloroformate, stirring for 30 minutes at-2 to-8 ℃;

6. dropwise adding the activated cortisol derivative solution into the dissolved G6PDH solution, and stirring at 2-8 ℃ overnight;

7. purifying the solution in step 6 by G-25 gel chromatography column to obtain final product glucose-6-phosphate dehydrogenase-hapten conjugate, and storing at 2-8 deg.C.

Example 3 preparation of the kit

A kit for the detection of cortisol is prepared comprising:

reagent R1, comprising:

50mM HEPES，pH 7.0

10mM glucose-6-phosphate

10mM β -Nicotinamide adenine dinucleotide

250ng/ml Cortisol antibody (commercially available antibody, without particular limitation)

1 g/L bovine serum albumin

1g/L Tween20

1 g/L sodium azide;

reagent R2, comprising:

200mM Tris buffer, pH 8.0

G6 PDH-cortisol conjugate at 0.1. mu.g/ml

1 g/L bovine serum albumin

1g/L Tween 20

1 g/L sodium azide;

calibration products: 20mM HEPES buffer, and 0.0, 50.0, 100.0, 200.0, 400.0, 800.0ng/ml cortisol (or added as needed);

quality control product: 20mM HEPES buffer, and 120ng/ml, 260ng/ml, 440ng/ml cortisol (or added as needed).

And assembling the reagents (optionally containing quality control products and calibration products) into a cortisol 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]
WAVE(SUB/MAIN)	[410][340]
		S.VIL.	[4.0]
S.R1	[100]
		S.R3	[100]
ABS.LIMIT:	[32000][ increasing by one]
		CALIB TYPE:	[Spline]
POINT:	[6]SPAN PONIT[6]
		Calibration article	0.0、50.0、100.0、200.0、400.0、800.0ng/ml
Sample(s)	The sample to be tested is various physiological samples, such as serum and plasma

Detection example 1 accuracy, precision, and linearity experiment of the kit of the present application

TABLE 2 accuracy, precision (for the D306C mutant)

TABLE 3 linearity

	Side 1	Side 2	Side 3	Mean value	Theoretical value	Relative deviation of	Absolute deviation
								1	5.9	7.6	4.0	5.8	1.53		4.30
2	74.3	73.3	75.5	74.4	72.04	3.2％	2.32
								3	146.5	145.2	142.6	144.8	142.55	1.6％	2.21
4	217.6	212.7	207.2	212.5	213.06	-0.3％	-0.56
								5	283.2	282.0	282.8	282.7	283.57	-0.3％	-0.91
6	344.9	344.1	346.0	345.0	354.08	-2.6％	-9.08
								7	408.9	421.3	421.9	417.4	424.59	-1.7％	-7.23
8	489.1	488.3	504.6	494.0	495.11	-0.2％	-1.11
								9	562.9	557.7	579.2	566.6	565.62	0.2％	0.98
10	627.9	655.4	646.6	643.3	636.13	1.1％	7.17
								11	707.0	695.9	723.2	708.7	706.64	0.3％	2.06
12	782.3	777.9	759.0	773.1	777.15	-0.5％	-4.08
								13	835.8	858.3	860.6	851.6	847.66	0.5％	3.91

Detection example 2 anti-interference of common drugs

TABLE 4 determination of interference resistance (against D306C mutant)

Detection example 3 correlation

1. Test method

100 samples of fresh serum were taken, each divided into two aliquots, each at a volume of not less than 500. mu.l, and one of the samples was assayed twice on Hitachi 7180 apparatus using the reagents of the present application (for the 375 mutant) and the other sample was assayed using Shimadzu HP L C. the measurements of the two methods were correlated using a scatter plot.

2. And (3) test results:

the resulting function is y 1.011x +1.9381, correlation coefficient R²＝0.9970。

The results show that the reagent of the present application measures cortisol concentration values in samples, and has good correlation with the method HP L C (which can be regarded as gold standard) for measuring cortisol concentration values in samples.

TABLE 5 correlation analysis (unit: ng/ml)

Test example 4 Interbatch Difference in Cortisol test kit

Three batches of the reagent of the present application (D306C mutant) and the reagent prepared by the control coupling method were used, and the difference in absorbance change between the different batches was calculated by separate calibration.

TABLE 6 calibration data between batches

TABLE 7 comparison between batches

Test example 5 calibration stability comparative analysis

The reagent of the present application (G426C mutant) and the reagent of the control coupling method were placed in a reagent chamber, uncapped (FIG. 4), and calibrated, three serum samples were measured three times each at regular intervals, and the measurement was continued for 14 days, and the mean and the deviation were calculated.

TABLE 8 calibration stability (unit: ng/ml)

Test example 6 antibody inhibition Rate

1. Detection principle of antibody inhibition rate

When the antibody is combined with the G6 PDH-cortisol conjugate, the activity of 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 experimental group in which the antibody is added and an experimental group in which the antibody is not added is compared by detecting the change of NADH amount, wherein the difference is represented by the inhibition capacity of the antibody on G6 PDH.

2. Reaction system

TABLE 9 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the added antibody with the unadditized antibody, and respectively detecting the absorbance values of the G6 PDH-cortisol conjugate to obtain the inhibition of the antibody on G6 PDH.

The antibody inhibition rate was defined as change in absorbance of G6 PDH-cortisol with antibody/change in absorbance of G6 PDH-cortisol 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 33 percent (G426C: 33 percent; D375C: 48 percent) and can reach 54 percent (D306C). Whereas the inhibition rates of previously published mutation sites (e.g. a45C, K55C) were 31% and 42%.

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 mutation site (i.e. the site for introducing free sulfydryl) in the enzyme mutant of the application is the site for coupling 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 cortisol is prepared into the kit, the reagent has obvious performance improvement in the aspects of inter-batch variation coefficient, linearity, specificity and the like.

Test example 7 alternative

Referring to the preparation method of example 3, different test kits and control kits were respectively configured, except that the kit prepared in example 3 was replaced as follows:

scheme 1: the buffer in the first reagent and the second reagent is replaced by phosphate buffer, glycine buffer, boric acid buffer, or MOPS buffer in the range of 50 to 100mM pH 7.0-8.0;

scheme 2 the stabilizer in the first reagent and the second reagent is replaced by 0.5 to 2.5 g/L trehalose, sucrose, mannitol, or polyethylene glycol 6000;

protocol 3 surfactant in the first and second reagents was replaced with 0.5 to 2.5 g/L Triton X-100, Tween80, Brij35, or Brij 23;

scheme 4: the preservatives in the first and second agents were replaced with lithium azide or PC-300.

Scheme 5: compound 2 was replaced with compound 3, compound 4, compound 5, respectively.

The test kit and the control kit of three different batches in each scheme are tested according to the method of the detection example 4, the comparison result is close to that in tables 6 to 7, and the difference variation between the batches of the test kit is smaller than that of the control kit (data not shown).

Sequence listing

<110> Beijing Jiuqiang Biotechnology Ltd

<120> 6-phosphoglucose dehydrogenase mutant and application thereof in preparing cortisol detection reagent

<130>390270CG

<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 (L eutonostoc pseudomesenteroides)

<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 150155 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 310315 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 GluAla 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 AlaAla 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 GlyAsp 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

3540 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 200205

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 360365

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 AlaAla 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 of a glucose-6-phosphate dehydrogenase mutant and a hapten in a molar ratio of 1: 1 is coupled;

the hapten is cortisol or a derivative thereof;

preferably, the cortisol derivative is of formula I:

wherein the content of the first and second substances,

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 and SEQ ID No. 4.

3. The conjugate of claim 1 or 2, wherein the cortisol derivative has a structure selected from the following formulae:

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, wherein:

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

the detection device is a cortisol detection device;

the detection device is selected from any one of the following forms: pore plate, particle, chip and test paper;

preferably, the test device is used for homogeneous immunoassay of cortisol.

7. A cortisol detection kit comprising:

a first reagent comprising a substrate, a cortisol 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/m L to 800ng/m L cortisol, and

optionally, a quality control comprising 10mM to 500mM buffer, 100ng/m L to 500ng/m L cortisol.

8. The cortisol detection kit according to claim 7, which includes:

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 β -nicotinamide adenine dinucleotide,

10ng/ml to 10. mu.g/ml, preferably 100ng/ml to 500ng/ml, of a cortisol antibody,

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

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

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

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

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

the buffer is selected from one or a combination of the following: phosphate buffer, glycine buffer, Tris buffer, boric acid buffer, MOPS buffer and HEPES buffer;

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: brij23, 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, biological preservative PC, thimerosal;

preferably, the preservative is selected from the group consisting of: sodium azide, lithium azide and PC-300.

9. The cortisol detection kit according to claim 7, which includes:

a first reagent comprising:

50mM HEPES，pH 7.0、

10mM glucose-6-phosphate,

10mM oxidized β -nicotinamide adenine dinucleotide,

250ng/ml cortisol antibody,

1 g/L bovine serum albumin,

1g/L Tween20、

1 g/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,

1 g/L bovine serum albumin,

1g/L Tween20、

1 g/L sodium azide.

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

1) providing cortisol or a derivative thereof;

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

3) the glucose-6-phosphate dehydrogenase mutant is coupled with the cortisol or the derivative thereof;

the cortisol derivative is of formula I:

wherein the content of the first and second substances,

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

preferably, the method for preparing the conjugate comprises the following steps:

1) providing a cortisol 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 cortisol derivative at 18 ℃ to 28 ℃ for 1 hour to 4 hours, preferably 2 hours to 3 hours, such that the cortisol derivative and the glucose-6-phosphate dehydrogenase mutant are coupled to obtain the conjugate;

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

step 1) and step 2) are interchangeable or parallel;

the buffer is selected from: PBS, Tris, TAPS, TAPSO,

the buffer pH 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 a free thiol group; more preferably, the glucose-6-phosphate dehydrogenase mutant has a free thiol group at position 306, 375 or 426.