CN116773827A

CN116773827A - Use of conjugates in the preparation of detection reagents

Info

Publication number: CN116773827A
Application number: CN202310724066.4A
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: 2023-09-19
Also published as: CN117054643A; CN116718764A; CN116124721A; CN111650135A; CN111537451A; CN112285037A; CN116338215A; CN116840467A; CN111504920A; CN116718761A; CN116626281A; CN116735512A; CN116148198A; CN117030640A; CN112285038A; CN111537452B; CN116359146A; CN116699122A; CN116773795A; CN111487207A

Abstract

The 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 relates to a division application of a Chinese patent application 6-phosphoglucose dehydrogenase mutant and application thereof in preparation of cortisol detection reagent (application number 2019114041546) filed on 12 months and 31 days 2019.

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 cortisol 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. Hapten, once bound to a protein, constitutes an antigenic cluster of the protein. Some chemically active substances (such as penicillin, sulfonamides, etc.) which have a smaller molecular weight than the general hapten but a specific structure are called simple haptens.

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.

Cortisol (cortison) has the structural formula:

cortisol, also known as hydrocortisone or compound F, is an adrenocortical hormone (which is one of the glucocorticoids) which is extracted from the adrenocortical tissue and has the strongest effect on carbohydrate metabolism.

Cortisol is produced from 11-deoxycortisol by the action of 11 beta-hydroxylase in the adrenal cortex mitochondria. Cortisol may also become a corticosteroid by the action of 11-beta-hydroxysteroid dehydrogenase (11-beta-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, immunomodulation, stress response, cell proliferation and differentiation, and memory regulation and cognitive function, among others. Cortisol in the blood binds mainly to the corticosteroid-binding globulins and albumin, with free cortisol only accounting for 3-5%, and cortisol concentration levels show periodic changes every day, with the lowest in the first half of the night and peak in the early morning.

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

Currently known cortisol detection methods mainly include: enzyme-linked immunosorbent assay, chemiluminescent immunoassay, high performance liquid chromatography, gas-liquid chromatography, gas chromatography and mass spectrometry. However, the detection methods have more defects, such as chemiluminescence, which has better sensitivity, but needs matched special equipment, so that the detection method has higher input and use cost and is not beneficial to popularization. In the clinical detection and diagnosis process, the homogeneous enzyme immune method (EMIT) and the latex enhanced turbidimetric immunoassay are mainly adopted.

Principle of homogeneous enzyme immunoassay: in a liquid homogeneous reaction system, enzyme-labeled antigen (such as G6 PDH-cortisol) and unlabeled antigen (cortisol) compete for being combined with quantitative antibody (cortisol antibody), when the more the antibody is combined with unlabeled antigen, the more the activity of the enzyme-labeled antigen is released, the more the NAD+ serving as a substrate is catalyzed by the enzyme to generate NADH, and the absorbance change of the NADH is detected at the wavelength of 340nm, so that the content of the cortisol in the liquid can be deduced.

The existing homogeneous enzyme immunoassay and latex agglutination turbidimetry are often limited in application due to complex preparation process and large batch-to-batch difference.

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

1) Dissolving G6PDH in Tris-containing MgCl at room temperature ₂ And NaCl (ph=9.0);

2) Adding NADH, glucose 6-phosphate and carbitol;

3) Then dimethyl sulfoxide is added dropwise;

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

5) Isobutyl chloroformate is added and stirred for 30 minutes at the temperature of-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 obtained in step 6) by means of a chromatographic column, the final product obtained is a conjugate of a G6 PDH-cortisol derivative (for example, but not limited to the method described in CN105131105 a).

However, the prior art methods rely on activation of reactive groups carried by the small molecule drug itself prior to reaction with the enzyme. Such strategies have difficulty guaranteeing orientation between small molecule drugs and enzymes 1:1, 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 cortisol detection kit.

According to some embodiments, a glucose 6-phosphate dehydrogenase mutant is provided. Unlike the mutants of glucose 6 phosphate dehydrogenase 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, 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, 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 application.

According to some embodiments, there is provided a host cell comprising an expression vector of the 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: and 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 molar ratio of glucose 6-phosphate dehydrogenase mutants of the present application to hapten is preferably 1:1.

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.

According to the present application, the skilled person will understand that "hapten" also includes the form of its derivative. In order to facilitate coupling with glucose-6-phosphate dehydrogenase, haptens (e.g., cortisol) 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: theophylline, phenytoin, vitamin D, 25 hydroxy vitamin D, 1, 25 dihydroxyvitamin D, folic acid, cardiac glycoside (including digitoxin), zymophenolic acid, lei Paming, cyclosporin A, amiodarone, methotrexate, tacrolimus, serum amino acids, bile acids, glycocholic acid, phenylalanine, ethanol, uronidine metabolite cotinine, uromorphine, uromonophenol derivatives, neuropeptide tyrosine, plasma galanin, polyamines, histamine, thyroid stimulating hormone, prolactin, placental lactation, growth hormone, follicle stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, antidiuretic hormone, calcitonin, procalcitonin, parathyroid hormone, thyroxine, triiodothyronine, anti-triiodothyronine, free thyroxine, free triiodothyronine, cortisol urine 17-hydroxycortic steroids, urine 17-ketosterols, dehydroepiandrosterone and sulfates, aldosterone, uronolamine 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-keto prostaglandin f1α, prostacyclin, epinephrine, catecholamine, norepinephrine, cholecystokinin, natriuretic acid adenosine cyclophosphate, cyclic guanosine monophosphate, vasoactive peptides, somatostatin, secretin, P-substance, neurotensin, thromboxane A2, thromboxane B2, 5 hydroxytryptamine, neuropeptide Y, osteocalcin.

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

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

In a specific 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, for example 1, 2, 3, 4, 5, 6.

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

The skilled artisan will appreciate that the X function is 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 an agent comprising a conjugate of the application.

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

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

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

5mM to 50mM substrate,

10ng/ml to 10. Mu.g/ml cortisol antibody,

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

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

0.1g/L to 5g/L preservative;

a second reagent comprising:

10mM to 500mM buffer,

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

0.1g/L to 5g/L of 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 the following: tromethamine buffer, phosphate buffer, tris-HCl buffer, citric acid-sodium citrate buffer, barbital buffer, glycine buffer, borate buffer, tris buffer; preferably, a phosphate buffer; the concentration of the buffer is 10mmol/L to 500mmol/L, preferably 100mM; the pH of the buffer is 7 to 8.

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 surfactant is selected from one or a combination of the following: brij35, triton X-100, triton X-405, tween20, tween30, tween80, coconut fatty acid diethanolamide, AEO7, preferably Tween20.

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 cortisol antibody is derived from: mice, rats, cats, dogs, primates, cows, horses, sheep, camelids, birds, humans.

In some specific embodiments, the cortisol 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 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, said buffer having a pH of 6.0 to 8.0);

3) At 18 ℃ to 28 ℃, the glucose 6-phosphate dehydrogenase mutant and the cortisol derivative are mixed according to a molar ratio of 1: n for 1 to 4 hours (preferably 2 to 3 hours) to allow coupling of the cortisol derivative and the glucose 6-phosphate dehydrogenase mutant to occur to give 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 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 enzyme to hapten in the reaction system is 1: n, where n is 0.01 to 1, e.g., 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) may 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 cortisol.

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. 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); is derived from Leuconostoc pseudomesenteroides Leuconostoc pseudomesenteroides.

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).

Figure 4. Decap stability.

Detailed Description

Examples

EXAMPLE 1 Synthesis of cortisol derivatives

Cortisol (200 mg,0.55 mmol), compound 2 (78 mg,2.20 mmol) was dissolved in 10ml methanol under dark conditions and reacted for 5 minutes under dark conditions.

The reaction system was heated to 50℃and to the reaction system were added Compound 2 (78 mg,2.20 mmol) and Compound 3 (60 mg,0.55 mmol), followed by reaction at a constant temperature for 5 minutes. The reaction system turns colorless or pale yellow basically, the solvent is removed under reduced pressure, and the product is obtained by column chromatography purification, 180mg, and the yield is 75%.

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

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

This example provides cortisol with a group that can bind to enzymes.

EXAMPLE 2 coupling of cortisol derivative to G6PDH molecule

1. The test method of the application

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

1. Preparing a solution:

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

g6pdh solution: g6pdh (mutant of the application or mutant of the prior art) was dissolved in PB 100mmol, naCl 100mmol, ph=8.0;

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

desalination solution: 100mM PB/K, 0.1% NaN ₃ 、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 hours.

3. After the reaction system was subjected to a shaking reaction at room temperature for 4 hours, the desalted solution was used to elute the reaction system using a desalting column, and the protein peak was collected to obtain a G6 PDH-cortisol conjugate.

2. Control coupling method (refer to CN105131105A method for making the experimental protocol)

1. 15mg of 100KU G6PDH was weighed and dissolved in 12mL of a solution containing 72.6mg (0.05M) Tris and 8mg MgCl at room temperature ₂ (3.3 mM) and 100mg NaCl (this solution ph=9.0);

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

3. 2mL of dimethyl sulfoxide is added dropwise into the beaker;

4. 10mg of cortisol derivative was weighed in anhydrous state and dissolved in 600. Mu.L of DMF; reducing the temperature of the solution to-2 to-8 ℃; 3. Mu.L tributylamine was added;

5. 1.5. Mu.L of isobutyl chloroformate was added and stirred 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. the solution in step 6 was purified by G-25 gel chromatography column and the final product obtained was glucose-6-phosphate dehydrogenase-hapten conjugate stored at 2 to 8 ℃.

EXAMPLE 3 preparation of the kit

The following kit for detecting cortisol was prepared, which comprises:

reagent R1 comprising:

50mM HEPES，pH 7.0

10mM glucose 6-phosphate

10mM beta-nicotinamide adenine dinucleotide

250ng/ml cortisol 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 μg/ml G6 PDH-cortisol conjugate

1g/L bovine serum albumin

1g/L Tween 20

1g/L sodium azide;

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

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

The reagent (optionally including quality control substances and calibration materials) is assembled into the cortisol homogeneous enzyme immunoassay kit.

Test case

TABLE 1 full automatic Biochemical instrument parameters

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][ incremental increase ]]
		CALIB TYPE:	[Spline]
POINT:	[6]SPAN PONIT[6]
		Calibration material	0.0、50.0、100.0、200.0、400.0、800.0ng/ml
Sample of	The sample to be detected is various physiological samples such as serum and plasma

Detection example 1. Accuracy, precision and linearity experiments of the kit of the present application

TABLE 2 accuracy and precision (for D306C mutant)

TABLE 3 linearities

	Measurement 1	Measurement 2	Measurement 3	Mean value of	Theoretical value	Relative deviation of	Absolute deviation of
								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 measurement results of anti-interference (for D306C mutant)

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Detection example 3 correlation

1. Test method

100 fresh serum samples were taken, each divided into two equal parts, each at a volume of not less than 500 μl, one of which was measured twice on a Hitachi 7180 instrument using the reagent of the present application (for 375 mutant) and the other was measured using Shimadzu HPLC. Both methods measure correlation analysis using scatter plots.

2. Test results:

the resulting function is y=1.01dx+1.9381, correlation coefficient r2=0.9970.

The results show that the cortisol concentration value in the sample measured by the reagent provided by the application has good correlation with the cortisol concentration value in the sample measured by an HPLC method (which can be regarded as a gold standard).

TABLE 5 correlation analysis (Unit: ng/ml)

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Detection example 4 differential between batches of cortisol detection kit

Three batches of the reagent (D306C mutant) of the application and the reagent prepared by the control coupling method were used for calibration respectively, and the absorbance variation difference of different batches was calculated.

TABLE 6 calibration data between batches

TABLE 7 comparison between batches

Test example 5 comparative analysis of calibration stability

The reagent (G426C mutant) and the reagent of the control coupling method are respectively placed in a reagent bin, uncapped (figure 4) and calibrated, three levels of serum samples are measured three times at intervals, the measurement is continued for 14 days, and the mean value and the deviation are calculated.

TABLE 8 calibration stability (Unit: ng/ml)

Detection example 6 antibody inhibition Rate

1. Principle of detection of antibody inhibition

When the antibody is combined with the G6 PDH-cortisol 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 experiment group to which the antibody is added and an experiment group to which the antibody is not added is compared by detecting the change of the NADH amount, and the difference is expressed as the inhibition capability of the antibody on the G6 PDH.

2. Reaction system

TABLE 9 preparation of reagents for detection of antibody inhibition

3. Results

And comparing the absorbance measurement value of the G6 PDH-cortisol 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.

Antibody inhibition = 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 obvious improvement on the antibody inhibition rate, can reach more than 33% (G426C: 33%; D375C: 48%), and can reach up to 54% (D306C). Whereas the inhibition rates of the previously published mutation sites (e.g.A45C, K55C) were 31% and 42%.

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 the greatest effect 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 the cortisol is prepared into a kit, the reagent has obvious performance improvement in the aspects of the 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 prepared, except that the kit prepared in example 3 was replaced as follows:

scheme 1: the buffer in the first and second reagents is replaced with phosphate buffer, glycine buffer, boric acid buffer, or MOPS buffer at a PH in the range of 50 to 100mm 7.0-8.0;

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

scheme 3: the surfactant in the first and second reagents is replaced with 0.5 to 2.5g/LTriton X-100, tween80, brij35, or Brij 23;

scheme 4: the preservative in the first and second reagents is replaced with lithium azide or PC-300.

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

Test kits and control kits of three different batches in each of the above schemes were tested with reference to the method of test example 4, and the comparison results were close to tables 6 to 7, showing that the variation in the batch-to-batch difference of the test kit was smaller than that of the control kit (data not shown).

Claims

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

the detection reagent is a homogeneous enzyme immunoassay detection reagent of cortisol;

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

the cortisol derivative is represented by formula I:

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

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 any one of the following: maleimide, bromoacetyl, vinyl sulfone, aziridine; more preferably, X is maleimide;

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.