CN110003300B - Derivative of 17-hydroxysteroid, detection reagent and preparation method - Google Patents
Derivative of 17-hydroxysteroid, detection reagent and preparation method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J7/00—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms
- C07J7/008—Normal steroids containing carbon, hydrogen, halogen or oxygen substituted in position 17 beta by a chain of two carbon atoms substituted in position 21
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
Abstract
The invention discloses a derivative of 17-hydroxysteroid, a detection reagent and a preparation method, and relates to the technical field of biological detection. The 17-hydroxysteroid immunogen prepared by the 17-hydroxysteroid derivative has high immunogenicity, and the obtained antibody has strong specificity and high titer; the 17-hydroxysteroid enzyme-labeled conjugate in the homogeneous enzyme immunoassay reagent prepared by the derivative is connected with recombinant glucose-6-phosphate dehydrogenase which is modified by genetic engineering, so that the detection sensitivity is obviously improved, a sample with the concentration as low as 80nmol/L can be effectively detected, the specificity is strong, and the reagent has no cross reaction with 92 common interferents; the method can realize high-flux and rapid detection of the content of the 17-hydroxysteroid on a full-automatic biochemical analyzer, has stable detection result, high accuracy and simple detection method, and is easy to realize, popularize and use.
Description
Technical Field
The invention relates to the technical field of biological detection, and particularly relates to a derivative of 17-hydroxysteroid, a detection reagent and a preparation method.
Background
17-hydroxysteroids (17-hydorxymycosteroids, 17-OHCS) having the formula (iv):
17-hydroxysteroids belong to the carbohydrate adrenocortical hormone, are hormones secreted by the adrenal cortex and metabolites thereof, mainly comprise cortisol, cortin, tetrahydrocortisol, tetrahydrocortin and the like, and are usually discharged out of the body along with urine. Therefore, the content of the 17-hydroxysteroid in urine can reflect the condition that the adrenal cortex secretes the cortisol, and can assist in diagnosing related endocrine diseases. The content reduction of 17-hydroxysteroid is commonly seen in adrenal cortex hypofunction, adenohypophysis hypofunction, bilateral adrenal gland resection, malnutrition, cirrhosis, tuberculosis, certain chronic consumptive diseases and the like; elevated levels of 17-hydroxysteroids are common in hypercorticism (caused by tumors or hyperplasia), ectopic ACTH syndrome, obesity, hyperthyroidism, pancreatitis, and the like.
The most commonly used method for detecting 17-hydroxysteroids in urine at present is a colorimetric assay. The principle of the method is as follows: under the acidic condition, 17-hydroxysteroid reacts with phenylhydrazine hydrochloride to generate yellow phenylhydrazine derivatives, the background color generated by sulfuric acid and other non-17-hydroxysteroid chromogenic substances is subtracted from the chromogenic result, and the content of the yellow phenylhydrazine derivatives can be obtained by processing and colorimetric the same as the standard solution. The method comprises the following detection steps: collecting urine to be detected; extracting a 17-hydroxysteroid sample; preparing a colorimetric assay sample; carrying out color development reaction; and (4) carrying out color comparison, reading the absorbance, and calculating the content of the 17-hydroxysteroid according to the absorbance. The method needs chloroform-n-butanol mixed solution for extraction, the reagent has anesthesia effect and irritation, and can be oxidized into hydrogen chloride and virulent phosgene under the action of light, thereby bringing harm to operators and environment; on the other hand, the method has the defects of poor stability of detection results, complex operation, long time consumption and unsuitability for large-scale clinical application.
With the continuous development of biological detection technology, the detection method of 17-hydroxysteroid is gradually improved. In the prior art, Chinese patent CN103335969B discloses a colorimetric determination method of 17-hydroxycorticosteroid, which does not contain toxic reagents such as trichloromethane, chloroform and the like, has safe and reliable operation process, does not need to use anhydrous ammonium sulfate and anhydrous sodium sulfate, and saves the reagents; the traditional extracted standby test solution is improved into the test solution which is directly used after pigment is removed, so that the step of preparing the standby test solution from the detected urine is greatly simplified, and the determination method is simple and easy to operate. Chinese patent CN105092831A discloses a 17-hydroxycorticosteroid immunodetection reagent and a preparation method thereof, wherein the reagent comprises an anti-17-hydroxycorticosteroid specific antibody and an indication reagent, and the minimum detection concentration of the reagent is 1 umol/L. At present, the amount of the detection reagent is less than that of the existing detection reagent on the market, and certain defects still exist, such as: poor stability, low sensitivity, and is not suitable for simultaneous detection of a large number of samples.
Therefore, aiming at the defects in the prior art, the research and development of a high-flux and rapid detection reagent which has the advantages of high sensitivity, good stability and accurate result and can be applied to a full-automatic biochemical analyzer has become a hotspot in the in vitro diagnostic reagent industry at home and abroad.
Disclosure of Invention
The invention aims to provide a derivative of 17-hydroxysteroid, a detection reagent and a preparation method. The homogeneous enzyme immunoassay reagent has the advantages of high sensitivity, good stability and strong specificity, and can realize full-automatic rapid detection of mass samples.
In one aspect, the invention provides a 17-hydroxysteroid derivative, wherein the structural formula of the 17-hydroxysteroid derivative is shown as the formula (I):
the invention also provides a preparation method of the 17-hydroxysteroid derivative, which comprises the following steps:
(1) synthesis of Compound 2: dissolving the compound 1 and triethylamine in dichloromethane, adding dihydrofuran-2, 5-dione, stirring, after the reaction is completed by thin-layer chromatography, quenching the reaction by using purified water, adjusting the pH value by using hydrochloric acid, extracting the dichloromethane for 3 times, combining organic layers, washing by using purified water and brine, and drying and concentrating in vacuum to obtain a white solid, namely a compound 2;
(2) synthesis of Compound 3: dissolving the compound 2 obtained in the step (1) in pyridine, and adding Pd (OH)2At room temperature and H2Stirring overnight under the conditions, filtering, vacuum concentrating, and purifying after TLC shows that all the starting materials are disappearedThe white solid, namely the compound 3 is obtained by the reaction;
(3) synthesis of 17-hydroxysteroid derivatives: dissolving the compound 3 obtained in the step (2) in anhydrous tetrahydrofuran, adding potassium triisobutylborohydride at-78 ℃, stirring, and after the thin-layer chromatography shows that the reaction is completely finished, using saturated NH4Quenching reaction with Cl aqueous solution, extracting with ethyl acetate for 3 times, mixing organic layers, washing with purified water and brine, vacuum drying, concentrating, and purifying to obtain white solid, i.e. 17-hydroxysteroid derivative;
the synthetic route of the 17-hydroxysteroid derivative is as follows:
specifically, the preparation method of the 17-hydroxysteroid derivative comprises the following steps:
(1) synthesis of Compound 2: dissolving 5g of compound 1 and 2.1g of Triethylamine (TEA) together in Dichloromethane (DCM) (50mL), then adding 1.8g of dihydrofuran-2, 5-dione in one portion at room temperature, stirring at room temperature for 2 hours, after completion of the reaction by Thin Layer Chromatography (TLC), quenching the reaction with 300mL of purified water, adjusting pH to 3 with 1N hydrochloric acid, extracting the reacted mixture with 300mL of DCM, repeating 3 times, combining the organic layers, washing with purified water (200mL) and brine (200mL), drying and concentrating in vacuo to obtain a white solid, i.e., compound 2;
(2) synthesis of Compound 3: compound 2 from step (1) was dissolved in pyridine (120mL), followed by addition of 4g of Pd (OH)2At room temperature and H2Stirring overnight under the condition, filtering the obtained solution after the thin-layer chromatography shows that all the initial reaction raw materials disappear, concentrating the filtrate in vacuum, and purifying the obtained crude product by silica gel to obtain a white solid, namely a compound 3;
(3) synthesis of 17-hydroxysteroid derivatives: dissolving the compound 3 obtained in the step (2) in anhydrous Tetrahydrofuran (THF) (50mL), dropwise adding potassium triisobutylborohydride (15mL, 1M) at-78 ℃, stirring at-78 ℃ for 1 hour, and after the reaction is completely finished by thin layer chromatography, using saturated NH4The reaction was quenched with aqueous Cl solution and then extracted with Ethyl Acetate (EA) (200mL) and repeated 3 times, the organic layers were combined, washed with purified water (200mL) and brine (200mL), dried and concentrated in vacuo, and the resulting crude product was purified on silica gel to give a white solid, the 17-hydroxysteroid derivative.
The invention also provides a 17-hydroxysteroid detection reagent, wherein the 17-hydroxysteroid detection reagent comprises a reagent A and a reagent B;
the reagent A comprises an anti-17-hydroxysteroid specific antibody and a homogeneous enzyme substrate;
the reagent B comprises a 17-hydroxysteroid enzyme-labeled conjugate and a Tris buffer solution;
the anti-17-hydroxysteroid specific antibody is a complete antibody molecule generated after a 17-hydroxysteroid immunogen immunizes an experimental animal, or an antibody fragment or an antibody derivative which retains the specific binding capacity with 17-hydroxysteroid;
the experimental animal is a mammal; preferably, the experimental animal is one of sheep, goat, mouse, guinea pig, rabbit or horse; more preferably, the experimental animal is a sheep.
The 17-hydroxysteroid immunogen is a compound obtained by connecting the 17-hydroxysteroid derivative or the 17-hydroxysteroid derivative prepared by the preparation method with a carrier, and the structural formula of the compound is shown as the formula (II):
the carrier is protein or polypeptide with immunogenicity;
preferably, the carrier is one of thyroglobulin, serum protein or hemocyanin; more preferably, the carrier is thyroglobulin;
the homogeneous enzyme substrate is prepared from glucose-6-phosphate, oxidized nicotinamide adenine dinucleotide and Tris buffer solution;
the 17-hydroxysteroid enzyme-labeled conjugate is formed by connecting the 17-hydroxysteroid derivative or the 17-hydroxysteroid derivative prepared by the preparation method with recombinant glucose-6-phosphate dehydrogenase (rG6PDH), and the structural formula is shown as the formula (III):
specifically, the amino acid sequence of the recombinant glucose-6-phosphate dehydrogenase (rG6PDH) is shown as SEQ ID NO: 1.
Preferably, the preparation method of the 17-hydroxysteroid immunogen comprises the following steps:
(A1) preparation of the carrier solution: dissolving a carrier in a phosphate buffer solution to obtain a carrier solution;
(A2) preparation of 17-hydroxysteroid derivative solution: mixing the 17-hydroxysteroid derivative, dimethylformamide, ethanol, a potassium phosphate buffer solution, 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and N-hydroxy thiosuccinimide, stirring for dissolving and reacting for 90min to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the carrier solution obtained in the step (A1), stirring overnight at 4 ℃, and dialyzing to purify the mixture to obtain the 17-hydroxysteroid immunogen.
Specifically, the preparation method of the 17-hydroxysteroid immunogen comprises the following steps:
(A1) preparation of the carrier solution: dissolving carrier protein in 0.2M phosphate buffer solution with pH of 8.5 to obtain carrier solution with carrier protein final concentration of 4 mg/mL;
(A2) preparation of 17-hydroxysteroid derivative solution: mixing 100-300mg of the 17-hydroxysteroid derivative, 1.75-5.25mL of dimethylformamide, 1.75-5.25mL of ethanol, 3.5-10.5mL of 10mM potassium phosphate buffer solution with pH of 5.0, 100-300mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 25-75mg of N-hydroxythiosuccinimide, and stirring for dissolving reaction for 30-60min to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the carrier solution obtained in the step (A1), stirring overnight at 4 ℃, and dialyzing to purify the mixture to obtain the 17-hydroxysteroid immunogen.
Preferably, the preparation method of the anti-17-hydroxysteroid specific antibody comprises the following steps:
(B1) diluting the 17-hydroxysteroid immunogen with PBS to obtain an antigen solution, mixing the antigen solution with an equivalent amount of Freund's complete adjuvant, and injecting the test animal;
(B2) after 5-6 weeks, injecting the experimental animal with the same antigen solution and equivalent Freund's incomplete adjuvant, and then injecting the experimental animal every 2 weeks for 5-8 times;
(B3) and (D) taking blood from the experimental animal obtained in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody.
Specifically, the preparation method of the anti-17-hydroxysteroid specific antibody comprises the following steps:
(B1) diluting the 17-hydroxysteroid immunogen to 0.1-3.0mg/mL by PBS to obtain an antigen solution, mixing the antigen solution with an equivalent amount of Freund's complete adjuvant, and injecting the experimental animal;
(B2) after 5-6 weeks, injecting the experimental animal with the same antigen solution and equivalent Freund's incomplete adjuvant, and then injecting the experimental animal every 2 weeks for 5-8 times;
(B3) and (D) taking blood from the experimental animal obtained in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody.
Preferably, the preparation method of the 17-hydroxysteroid enzyme-labeled conjugate comprises the following steps:
(C1) preparation of recombinant glucose-6-phosphate dehydrogenase solution: recombinant glucose-6-phosphate dehydrogenase, Tris buffer and MgCl2Mixing and dissolving with NaCl; then adding reduced nicotinamide adenine dinucleotide, glucose-6-phosphate, carbitol and dimethyl sulfoxide to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: dissolving the 17-hydroxysteroid derivative in dimethylformamide, cooling to-18 ℃, adding tributylamine and isobutyl chloroformate, and uniformly stirring at low temperature to obtain a 17-hydroxysteroid derivative solution;
(C3) obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight at 2-8 ℃, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
Specifically, the preparation method of the 17-hydroxysteroid enzyme-labeled conjugate comprises the following steps:
(C1) preparation of recombinant glucose-6-phosphate dehydrogenase solution: 7.5-22.5mg of recombinant glucose-6-phosphate dehydrogenase, 6-18mL of Tris buffer, 8mg of MgCl2Mixing with 100mg NaCl for dissolving; then adding 112.5-337.5mg of reduced nicotinamide adenine dinucleotide, 67.5-202.5mg of glucose-6-phosphate, 0.375-1.125mL of carbitol and 1-3mL of dimethyl sulfoxide to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: dissolving 5-15mg of the 17-hydroxysteroid derivative in 900 mul of dimethylformamide 300-;
(C3) obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight at 2-8 ℃, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
The invention also provides a preparation method of the 17-hydroxysteroid detection reagent, which comprises the following steps:
(D1) preparation of homogeneous enzyme substrate: dissolving oxidized nicotinamide adenine dinucleotide and glucose-6-phosphate in a Tris buffer solution according to the ratio of the final concentration to the final concentration of 1:1 to prepare a homogeneous enzyme substrate;
(D2) preparation of reagent A: uniformly mixing the homogeneous enzyme substrate obtained in the step (D1) with the anti-17-hydroxysteroid specific antibody to obtain a reagent A, wherein the volume ratio of the anti-17-hydroxysteroid specific antibody to the homogeneous enzyme substrate in the reagent A is 1: 50-5000;
(D3) preparation of reagent B: and dissolving the 17-hydroxysteroid enzyme-labeled conjugate in a Tris buffer solution to obtain a reagent B, wherein the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer solution in the reagent B is 1: 50-5000.
Preferably, the volume ratio of the anti-17-hydroxysteroid specific antibody to the homogeneous enzyme substrate in the reagent A is 1: 500; the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer solution in the reagent B is 1: 750.
The invention has the beneficial effects that:
the invention provides a derivative of 17-hydroxysteroid, a detection reagent and a preparation method. The antibody prepared from the derivative has strong specificity and high titer, and the 17-hydroxysteroid enzyme-labeled conjugate in the homogeneous enzyme immunoassay reagent prepared from the derivative is connected with recombinant glucose-6-phosphate dehydrogenase (rG6PDH) modified by genetic engineering, so that the detection sensitivity is remarkably improved, and a sample with the concentration as low as 80nmol/L can be effectively detected; the method can realize high-flux and rapid detection of the content of the 17-hydroxysteroid on a full-automatic biochemical analyzer, has high detection stability and accuracy, strong specificity, no cross reaction with 92 common interferents, obviously improved detection efficiency, simple detection method and easy realization, popularization and use.
Drawings
FIG. 1 is a standard curve of ELISA detection of 17-hydroxysteroids in example 5 of the present invention;
FIG. 2 is a homogeneous enzyme immunization standard curve for 17-hydroxysteroids of example 12 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1: synthesis of 17-hydroxysteroid derivatives
The synthetic route for the 17-hydroxysteroid derivatives is as follows:
the preparation method of the 17-hydroxysteroid derivative comprises the following specific steps:
(1) synthesis of Compound 2: 5g of compound 1 and 2.1g of Triethylamine (TEA) were dissolved together in Dichloromethane (DCM) (50mL), then 1.8g of dihydrofuran-2, 5-dione were added in one portion at room temperature, stirred at room temperature for 2 hours, after completion of the reaction by Thin Layer Chromatography (TLC), the reaction was quenched with 300mL of purified water, pH was adjusted to 3 with 1N hydrochloric acid, the mixture after the reaction was extracted with 300mL of DCM, repeated 3 times, the organic layers were combined, washed with purified water (200mL) and brine (200mL), dried and concentrated in vacuo to give 6g of a white solid, compound 2, yield: 94 percent;
(2) synthesis of Compound 3: compound 2(6g) obtained in step (1) was dissolved in pyridine (120mL), followed by addition of Pd (OH)2(4g) At room temperature and H2After stirring overnight under conditions and thin layer chromatography showed complete disappearance of starting material, the resulting solution was filtered and the filtrate was concentrated in vacuo to give the crude product which was purified over silica gel to give 3.5g of a white solid, compound 3, yield: 58.3 percent;
(3)17-synthesis of hydroxysteroid derivatives: compound 3(3.5g) from step (2) was dissolved in anhydrous Tetrahydrofuran (THF) (50mL), potassium triisobutylborohydride (15mL, 1M) was added dropwise at-78 deg.C, stirred at-78 deg.C for 1 hour, and after completion of the reaction by thin layer chromatography, saturated NH was used4The reaction was quenched with aqueous Cl solution and then extracted with Ethyl Acetate (EA) (200mL), repeated 3 times, the organic layers were combined, washed with purified water (200mL) and brine (200mL), dried and concentrated in vacuo to give the crude product which was purified on silica gel to give 1.1g of a white solid, yield: 31.4 percent.
And (3) performing nuclear magnetic resonance spectrum scanning on the white solid compound obtained in the step (3) by using Bruker Avance III plus 400MHz and VARIAN MERCURY plus 300M, and identifying the chemical structure by using TMS as an internal standard. The results are as follows:
1H NMR(CD3OD,400MHz):0.54-0.55(s,3H),1.54(s,1H),1.58-1.60(s,3H),1.64-1.68(m,4H),1.74-1.75(m,2H),1.80-1.81(m,2H),1.82-1.83(m,2H),1.9-2.1(m,5H),2.26-2.27(m,1H),2.50-2.75(m,6H),2.90-3.10(m,1H),3.90-4.10(d,1H),4.85-4.90(d,1H),4.95-5.10(d,1H).
the structural formula is shown as the formula (I):
the results indicated that the white solid was a 17-hydroxysteroid derivative.
Example 2: preparation of 17-hydroxysteroid immunogens and anti-17-hydroxysteroid specific antibodies
The preparation steps of the 17-hydroxysteroid immunogen are as follows:
(A1) preparation of hemocyanin solution: dissolving 100mg of hemocyanin in 25mL of 0.2M phosphate buffer solution with the pH value of 8.5 to obtain a hemocyanin solution;
(A2) preparation of 17-hydroxysteroid derivative solution: 100mg of the 17-hydroxysteroid derivative obtained in example 1, 1.75mL of dimethylformamide, 1.75mL of ethanol, 3.5mL of 10mM potassium phosphate buffer solution having a pH of 5.0, 100mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide, and 25mg of N-hydroxythiosuccinimide were mixed and dissolved with stirring for 90 minutes to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the hemocyanin solution obtained in the step (A1), stirring overnight at 4 ℃, and dialyzing for purification to obtain the 17-hydroxysteroid immunogen.
The anti-17-hydroxysteroid specific antibody was prepared as follows:
(B1) diluting the 17-hydroxysteroid immunogen to 0.1mg/mL by PBS to obtain an antigen solution, mixing the antigen solution with an equivalent amount of Freund's complete adjuvant, and injecting the goat experimental animal;
(B2) after 5 weeks, injecting the goat experimental animal with the same antigen solution and an equivalent amount of Freund's incomplete adjuvant, and then injecting the goat experimental animal once every 2 weeks for 5 times in total;
(B3) and (4) taking blood from the experimental animal goat in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody with the titer of 1: 30000.
Example 3: preparation of 17-hydroxysteroid immunogens and anti-17-hydroxysteroid specific antibodies
The preparation steps of the 17-hydroxysteroid immunogen are as follows:
(A1) preparation of thyroglobulin solution: dissolving 200mg of thyroglobulin in 50mL of 0.2M phosphate buffer solution with the pH of 8.5 to obtain a thyroglobulin solution;
(A2) preparation of 17-hydroxysteroid derivative solution: 200mg of the 17-hydroxysteroid derivative obtained in example 1, 3.5mL of dimethylformamide, 3.5mL of ethanol, 7.0mL of 10mM potassium phosphate buffer solution having a pH of 5.0, 200mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 50mg of N-hydroxythiosuccinimide were mixed and dissolved with stirring for 90 minutes to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the thyroglobulin solution obtained in the step (A1), stirring overnight at 4 ℃, and purifying by dialysis to obtain the 17-hydroxysteroid immunogen.
The anti-17-hydroxysteroid specific antibody was prepared as follows:
(B1) diluting the 17-hydroxysteroid immunogen to 1.0mg/mL by PBS to obtain an antigen solution, mixing the antigen solution with an equal amount of Freund's complete adjuvant, and injecting the experimental animal sheep;
(B2) after 5 weeks, injecting the sheep of the experimental animal with the same antigen solution and an equivalent amount of Freund's incomplete adjuvant, and then injecting the sheep once every 2 weeks for 6 times in total;
(B3) and (D) taking blood from the experimental animal sheep in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody with the titer of 1: 50000.
Example 4: preparation of 17-hydroxysteroid immunogens and anti-17-hydroxysteroid specific antibodies
The preparation steps of the 17-hydroxysteroid immunogen are as follows:
(A1) preparation of serum protein solution: dissolving 300mg of serum protein in 75mL of 0.2M phosphate buffer at pH 8.5 to obtain a serum protein solution;
(A2) preparation of 17-hydroxysteroid derivative solution: 300mg of the 17-hydroxysteroid derivative obtained in example 1, 5.25mL of dimethylformamide, 5.25mL of ethanol, 10.5mL of 10mM potassium phosphate buffer solution having a pH of 5.0, 300mg of 1-ethyl-3- (-3-dimethylaminopropyl) carbodiimide and 75mg of N-hydroxythiosuccinimide were mixed and dissolved with stirring for 90 minutes to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the serum protein solution obtained in the step (A1), stirring overnight at 4 ℃, and dialyzing to purify the mixture to obtain the 17-hydroxysteroid immunogen.
The anti-17-hydroxysteroid specific antibody was prepared as follows:
(B1) diluting the 17-hydroxysteroid immunogen to 3.0mg/mL by PBS to obtain an antigen solution, mixing the antigen solution with an equivalent amount of Freund's complete adjuvant, and injecting the experimental animal mouse;
(B2) after 6 weeks, injecting the same antigen solution and equivalent Freund's incomplete adjuvant into the experimental animal mice, and then injecting the antigen solution once every 2 weeks for 8 times in total;
(B3) and (D) taking blood from the experimental animal mouse in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody with the titer of 1: 40000.
Example 5: ELISA assay for 17-hydroxysteroids
(1) Establishing an ELISA detection standard curve of the 17-hydroxysteroid
Preparation of a standard substance: 17-hydroxysteroid powder (purchased from Sigma) was dissolved in methanol to prepare a stock solution of 1000. mu. mol/L. Stock solutions were diluted sequentially with ELISA buffer to 800.0, 400.0, 200.0, 100.0, 50.0, 0.0nmol/L standard solutions. Wherein, the ELISA buffer solution contains 50.0mM Tris, 145mM NaCl and 0.25% of Bovine Serum Albumin (BSA) by mass fraction.
Drawing a standard curve: the anti-17-hydroxysteroid specific antibody prepared in example 3 was diluted with PBS to 1: 8000 final concentration solution, 100 mul/well coated on 96-well enzyme-linked plate, 4 ℃ standing for 12-24h, PBS using PBS to wash the 96-well enzyme-linked plate coated with the antibody for 3 times, adding 200 mul/well BSA solution with mass fraction of 0.5%, 4 ℃ blocking and standing for 8-16h, PBS using PBS to wash for 3 times, adding 20 mul/well standard substance, adding 100 mul/well Horse Radish Peroxidase (HRP) -17-hydroxysteroid conjugate, incubating at room temperature for 30min, PBS washing 5 times, then adding 100 mul tetramethyl benzidine (TMB) substrate per well, incubating at room temperature for 30min, and adding 100 mul stop solution (2M sulfuric acid) per well. The absorbance was measured at a wavelength of 450nm to prepare a calibration curve, and the results are shown in FIG. 1.
(2) Detection of 17-hydroxysteroid content in sample to be detected
Preparation of a sample to be tested: 17-hydroxysteroid powder (purchased from Sigma) was dissolved in methanol solution to prepare a stock solution of 1000. mu. mol/L, and this stock solution was diluted in blank urine to a final concentration of 0.00, 80.00, 320.00, 800.00nmol/L, respectively, to form a blank, low, medium, and high concentration urine sample, which was urine of a healthy human without 17-hydroxysteroid.
Detecting a sample to be detected: and (3) using the detection method in the step (1) to replace the standard substance with blank, low, medium and high concentration urine samples, performing 3-time multi-well measurement on each sample, and detecting the light absorption value of the blank, low, medium and high concentration urine samples at 450 nm. The content of 17-hydroxysteroid in each sample was calculated from the standard curve shown in fig. 1, and the recovery rate, i.e., the detection concentration/sample concentration × 100%, was calculated from the actual content of 17-hydroxysteroid in the sample, and the results are shown in table 1.
Table 1: ELISA test results for 17-hydroxysteroids
| Urine sample | Blank space | Is low in | In | Height of |
| Sample concentration (nmol/L) | 0.00 | 80.00 | 320.00 | 800.00 |
| Test 1 | 0.01 | 81.25 | 319.66 | 811.35 |
| Test 2 | 0.03 | 80.41 | 315.58 | 808.20 |
| Test 3 | 0.02 | 80.56 | 317.73 | 805.44 |
| Average value (nmol/L) | 0.02 | 80.74 | 317.66 | 808.60 |
| Recovery (%) | - | 100.9 | 99.3 | 101.0 |
From the results in Table 1, it can be seen that: the 17-hydroxysteroid ELISA detection reagent provided by the invention is used for determining the recovery rate of 17-hydroxysteroid in samples with different concentrations, the recovery rate is higher and is less than or equal to 101% when the recovery rate is more than 99%, which indicates that the anti-17-hydroxysteroid specific antibody provided by the invention can be used for detecting 17-hydroxysteroid in samples, and the result accuracy is high and the stability is good.
In addition, the invention also carries out ELISA detection on the 17-hydroxysteroid specific antibody prepared in other examples, and the recovery rate of the 17-hydroxysteroid is higher and can reach more than 95 percent and less than 105 percent.
Example 6: preparation of 17-hydroxysteroid enzyme-labeled conjugate
(C1) Recombinant glucose-6-Preparation of phosphate dehydrogenase solution: 7.5mg of recombinant glucose-6-phosphate dehydrogenase (amino acid sequence shown in SEQ ID NO: 1), 6mL of Tris buffer, 8mg of MgCl2Mixing with 100mg NaCl for dissolving; then adding 112.5mg of reduced nicotinamide adenine dinucleotide, 67.5mg of glucose-6-phosphate, 0.375mL of carbitol and 1mL of dimethyl sulfoxide to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: dissolving 5mg of the 17-hydroxysteroid derivative obtained in example 1 in 300. mu.l of dimethylformamide, cooling to-18 ℃, adding 1.5. mu.l of tributylamine and 0.75. mu.l of isobutyl chloroformate, stirring at-2-8 ℃ for 30min, and uniformly mixing to obtain a 17-hydroxysteroid derivative solution;
(C3) obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight at 2-8 ℃, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
Example 7: preparation of 17-hydroxysteroid enzyme-labeled conjugate
(C1) Preparation of recombinant glucose-6-phosphate dehydrogenase solution: 15mg of recombinant glucose-6-phosphate dehydrogenase (amino acid sequence shown in SEQ ID NO: 1), 12mL of Tris buffer, 8mg of MgCl2Mixing with 100mg NaCl for dissolving; then 225mg of reduced nicotinamide adenine dinucleotide, 135mg of glucose-6-phosphate, 0.75mL of carbitol and 2mL of dimethyl sulfoxide are added to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: 10mg of the 17-hydroxysteroid derivative obtained in example 1 was dissolved in 600. mu.l of dimethylformamide, the temperature was reduced to-18 ℃ and 3. mu.l of tributylamine and 1.5. mu.l of isobutyl chloroformate were added, stirred at-2 to-8 ℃ for 30min and mixed to obtain a 17-hydroxysteroid derivative solution.
(C3) Obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight at 2-8 ℃, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
Example 8: preparation of 17-hydroxysteroid enzyme-labeled conjugate
(C1) Preparation of recombinant glucose-6-phosphate dehydrogenase solution: 22.5mg of recombinant glucose-6-phosphate dehydrogenase (amino acid sequence shown in SEQ ID NO: 1), 18mL of Tris buffer, 8mg of MgCl2Mixing with 100mg NaCl for dissolving; then 337.5mg of reduced nicotinamide adenine dinucleotide, 202.5mg of glucose-6-phosphate, 1.125mL of carbitol and 3mL of dimethyl sulfoxide are added to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: 15mg of the 17-hydroxysteroid derivative obtained in example 1 was dissolved in 900. mu.l of dimethylformamide, the temperature was reduced to-18 ℃ and 4.5. mu.l of tributylamine and 2.25. mu.l of isobutyl chloroformate were added, and the mixture was stirred at-2 ℃ to-8 ℃ for 30min and mixed to obtain a 17-hydroxysteroid derivative solution.
(C3) Obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight at 2-8 ℃, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
Example 9: preparation of 17-hydroxysteroid detection reagent
The method specifically comprises the following steps:
(D1) preparation of homogeneous enzyme substrate: dissolving 12.5G of Nicotinamide Adenine Dinucleotide (NAD) in an oxidized state and 5.2G of glucose-6-phosphate (G-6-P) in 3L80mM Tris buffer (pH 8.5) to obtain a homogeneous enzyme substrate, wherein the final concentration ratio of nicotinamide adenine dinucleotide in an oxidized state to glucose-6-phosphate is 1: 1;
(D2) preparation of reagent A: uniformly mixing the homogeneous enzyme substrate obtained in the step (D1) with the anti-17-hydroxysteroid specific antibody prepared in example 3 to obtain a reagent A, wherein the volume ratio of the anti-17-hydroxysteroid specific antibody to the homogeneous enzyme substrate in the reagent A is 1: 50;
(D3) preparation of reagent B: the 17-hydroxysteroid enzyme-labeled conjugate obtained in example 7 was dissolved in 100mM Tris buffer at pH 7.8 to obtain reagent B, in which the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to Tris buffer was 1: 50.
Example 10: preparation of 17-hydroxysteroid detection reagent
This example differs from example 9 in that the volume ratio of anti-17-hydroxysteroid specific antibody to homogeneous enzyme substrate in reagent A is 1: 500; the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer in the reagent B is 1: 750.
Example 11: preparation of 17-hydroxysteroid detection reagent
This example differs from example 9 in that the volume ratio of anti-17-hydroxysteroid specific antibody to homogeneous enzyme substrate in reagent A is 1: 5000; the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer solution in the reagent B is 1: 5000.
Example 12: homogeneous enzyme immunoassay for 17-hydroxysteroids
(1) Establishing a 17-hydroxysteroid homogeneous enzyme immunoassay standard curve
Preparation of a standard substance: the preparation method is the same as that of the ELISA detection standard in example 5.
Drawing a standard curve: the reaction parameters of the Mirey BS-480 full-automatic biochemical analyzer were set according to Table 2. The 17-hydroxysteroid homogeneous enzyme immunoassay reagent used was the detection reagent prepared in example 10. Adding the reagent A, adding the standard substance and finally adding the reagent B. After addition of reagent B, the OD was determined at various time points340And (3) calculating the light absorption value, calculating the reaction rate of different standard substance concentrations, and drawing a reaction standard curve, as shown in figure 2.
Table 2: merrill BS-480 full-automatic biochemical analyzer reaction parameter
| Name of item | 17-hydroxysteroids |
| Reagent A | 200μl |
| Reagent B | 50μl |
| Sample size | 12μl |
| Analytical method | End point method |
| Dominant wavelength | 340nm |
| Sub-wavelength | 412nm |
| Reaction time | 10 minutes |
| Incubation time | 5 minutes |
| Reaction direction | Rise up |
| Results | nmol/L |
| Accuracy of results | 0.01 |
| Calibration method | Logistic-Log 5P |
| Concentration of standard substance | 0.00，50.00，100.00，200.00，400.00，800.00nmol/L |
(2) Detecting a sample to be detected:
the sample to be tested is prepared by dissolving 17-hydroxysteroid standard substance in human urine to the concentration of 80.00, 320.00 and 800.00nmol/L respectively. The measurement of the low, medium and high concentration quality control samples was repeated 10 times, and the content and recovery rate of 17-hydroxysteroid in each sample were calculated from the standard curve shown in fig. 2, and the results are shown in table 3.
Table 3: sample determination and precision and recovery evaluation
| Urine sample | Is low in | In | Height of |
| Sample concentration (nmol/L) | 80.00 | 320.00 | 800.00 |
| 1 | 82.13 | 321.36 | 815.95 |
| 2 | 82.25 | 325.47 | 804.60 |
| 3 | 81.50 | 316.82 | 786.59 |
| 4 | 80.73 | 324.53 | 809.94 |
| 5 | 79.64 | 326.99 | 831.36 |
| 6 | 80.91 | 330.25 | 804.25 |
| 7 | 80.45 | 315.85 | 796.89 |
| 8 | 81.18 | 319.83 | 801.62 |
| 9 | 79.46 | 328.20 | 812.74 |
| 10 | 80.40 | 321.76 | 792.51 |
| Average value (nmol/L) | 80.84 | 323.16 | 805.65 |
| Standard Deviation (SD) | 0.095 | 0.485 | 1.281 |
| Precision (CV%) | 1.17 | 1.51 | 1.60 |
| The recovery rate is high | 101.1 | 100.9 | 100.7 |
And (3) detection results: the homogeneous enzyme immunoassay reagent has high determination accuracy, the recovery rate reaches 95-105%, the precision is high, and CV is lower than 3%.
Example 13 drug and hormone intervention test
Selecting 62 common drugs and 30 common hormones and hormone metabolites to carry out interference detection, adjusting the concentration of the interferents to be detected to 0.10 mu mol/L, and adopting the 17-hydroxysteroid homogeneous enzyme immunoassay method of the embodiment 12 to carry out detection.
The interferent to be detected is contacted and reacted with the reagent A prepared in the embodiment 10, and then the reagent B is added; the absorbance at 340nm was measured and the concentration of the corresponding substance was obtained according to the standard curve shown in FIG. 2. The names and the measurement results of 62 common drugs and 30 common hormones and hormone metabolites are shown in Table 4.
Table 4: results of homogeneous enzyme immunoassay for common interferents
The measurement results show that: the concentration of 17-hydroxysteroid equivalent to the concentration of the above 62 common drugs and 30 common hormones and hormone metabolites is less than 0.10 mu mol/L. Therefore, the 17-hydroxysteroid homogeneous enzyme immunoassay reagent provided by the invention has strong specificity and no cross reaction with 92 common interferents.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> Suzhou Boyuan medical science and technology Co., Ltd
<120> derivative of 17-hydroxysteroid, detection reagent and preparation method
<130>2019
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<170>SIPOSequenceListing 1.0
<210>1
<211>455
<212>PRT
<213> Artificial Synthesis (Artificial)
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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 Tyr Pro Ser Val Phe Asn Tyr Lys Lys Gly
20 25 30
Tyr Gln Lys His Phe Ala Ile Val Gly Thr Ala Arg Gln Ala Asn Asp
35 40 45
Asp Glu Phe Lys Gln Val Arg Asp Ser Ile Lys Asp Phe Thr Asp Asp
50 55 60
Gln Ala Gln Ala Glu Ala Phe Ile Glu His Phe Ser Tyr Arg Ala His
65 70 75 80
Asp Val Thr Asp Ala Ala Ser Tyr Ala Val Lys Glu Ala Ile Glu Glu
85 90 95
Ala Ala Asp Lys Phe Asp Ile Asp Gly Asn Arg Ile Phe Tyr Met Ser
100 105 110
Val Ala Pro Arg Phe Phe Gly Thr Ile Ala Lys Tyr Lys Ser Glu Gly
115 120 125
Ala Asp Thr Gly Tyr Asn Arg Met Ile Glu Lys Pro Phe Gly Thr Ser
130 135 140
Tyr Asp Thr Ala Ala Glu Gln Asn Asp Glu Asn Ala Phe Asp Asp Asn
145 150 155 160
Gln Phe Arg Ile Asp His Tyr Gly Lys Glu Met Val Gln Asn Ile Ala
165 170 175
Ala Arg Phe Gly Asn Pro Ile Phe Asp Ala Ala Trp Asn Lys Asp Tyr
180 185 190
Ile Lys Asn Val Gln Val Thr Ser Glu Val Gly Val Glu Glu Arg Ala
195 200 205
Gly Tyr Tyr Asp Thr Ala Gly Ala Asp Met Ile Gln Asn His Thr Met
210 215 220
Gln Ile Val Gly Trp Ala Met Glu Lys Pro Glu Ser Phe Thr Asp Lys
225 230 235 240
Asp Ile Arg Ala Ala Lys Asn Ala Ala Phe Asn Ala Lys Ile Tyr Asp
245 250 255
Glu Ala Glu Val Asn Lys Tyr Phe Val Arg Ala Gln Tyr Gly Ala Gly
260 265 270
Asp Ser Ala Asp Phe Lys Pro Tyr Glu Glu Asp Val Pro Ala Asp Ser
275 280 285
Lys Asn Asn Thr Phe Ile Ala Gly Glu Gln Phe Asp Pro Arg Trp Glu
290 295 300
Gly Val Pro Phe Tyr Val Arg Ser Gly Lys Arg Ala Ala Lys Gln Thr
305 310 315 320
Arg Val Asp Ile Val Phe Lys Ala Gly Thr Phe Asn Phe Gly Ser Glu
325 330 335
Gln Glu Ala Gln Glu Ala Val Ser Ile Ile Ile Asp Pro Lys Gly Ala
340 345 350
Ile Glu Lys Asn Ala Lys Ser Val Glu Asp Ala Phe Asn Thr Arg Thr
355 360 365
Ile Asp Gly Trp Thr Val Ser Asp Glu Asp Lys Lys Asn Thr Pro Glu
370 375 380
Pro Tyr Glu Arg Met Ile His Asp Thr Met Asn Gly Asp Gly Ser Asn
385 390 395 400
Phe Ala Asp Trp Asn Gly Val Ser Ile Ala Trp Lys Phe Val Asp Ala
405 410 415
Ile Ser Ala Val Tyr Thr Ala Asp Lys Ala Pro Glu Thr Tyr Lys Ser
420 425 430
Gly Ser Met Gly Pro Glu Ala Ser Asp Lys Leu Leu Ala Ala Asn Gly
435 440 445
Asp Ala Trp Val Phe Lys Gly
450 455
Claims (8)
1. A 17-hydroxysteroid detection reagent, wherein the 17-hydroxysteroid detection reagent comprises a reagent A and a reagent B;
the reagent A comprises an anti-17-hydroxysteroid specific antibody and a homogeneous enzyme substrate;
the reagent B comprises a 17-hydroxysteroid enzyme-labeled conjugate and a Tris buffer solution;
the anti-17-hydroxysteroid specific antibody is a complete antibody molecule generated after a 17-hydroxysteroid immunogen immunizes an experimental animal, or an antibody fragment or an antibody derivative which retains the specific binding capacity with 17-hydroxysteroid;
the experimental animal is a mammal;
the 17-hydroxysteroid immunogen is a compound obtained by connecting a 17-hydroxysteroid derivative shown in a formula (I) with a carrier, and the structural formula of the compound is shown in a formula (II):
the carrier is protein or polypeptide with immunogenicity;
the homogeneous enzyme substrate is prepared from glucose-6-phosphate, oxidized nicotinamide adenine dinucleotide and Tris buffer solution;
the 17-hydroxysteroid enzyme-labeled conjugate is formed by connecting a 17-hydroxysteroid derivative shown in the formula (I) and recombinant glucose-6-phosphate dehydrogenase, and the structural formula of the conjugate is shown in the formula (III):
the amino acid sequence of the recombinant glucose-6-phosphate dehydrogenase is SEQ ID NO: 1.
2. the reagent of claim 1, wherein the carrier is one of thyroglobulin, serum albumin or hemocyanin;
the experimental animal is one of sheep, goats, mice, guinea pigs, rabbits or horses.
3. The 17-hydroxysteroid detection reagent as defined in claim 2, wherein the carrier is thyroglobulin; the experimental animal is sheep.
4. The reagent for detecting 17-hydroxysteroid as set forth in claim 1, wherein the 17-hydroxysteroid immunogen is prepared by the method comprising the steps of:
(A1) preparation of the carrier solution: dissolving a carrier in a phosphate buffer solution to obtain a carrier solution;
(A2) preparation of 17-hydroxysteroid derivative solution: mixing the 17-hydroxysteroid derivative represented by the formula (i) in claim 1 with dimethylformamide, ethanol, potassium phosphate buffer, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxythiosuccinimide, and dissolving with stirring to obtain a 17-hydroxysteroid derivative solution;
(A3) obtaining a 17-hydroxysteroid immunogen: adding the 17-hydroxysteroid derivative solution obtained in the step (A2) into the carrier solution obtained in the step (A1), stirring overnight, and dialyzing to purify the mixture to obtain the 17-hydroxysteroid immunogen.
5. The reagent for detecting 17-hydroxysteroid according to claim 1, wherein the method for preparing the antibody specific to the anti-17-hydroxysteroid comprises the following steps:
(B1) diluting the 17-hydroxysteroid immunogen of claim 1 with PBS to obtain an antigen solution, mixing the antigen solution with an equal amount of Freund's complete adjuvant, and injecting into experimental animals;
(B2) after 5-6 weeks, injecting the experimental animal with the same antigen solution and equivalent Freund's incomplete adjuvant, and then injecting the experimental animal every 2 weeks for 5-8 times;
(B3) and (D) taking blood from the experimental animal obtained in the step (B2), separating and purifying to obtain the anti-17-hydroxysteroid specific antibody.
6. The reagent for detecting 17-hydroxysteroid as claimed in claim 1, wherein the preparation method of the enzyme-labeled conjugate for 17-hydroxysteroid comprises the following steps:
(C1) preparation of recombinant glucose-6-phosphate dehydrogenase solution: recombinant glucose-6-phosphate dehydrogenase, Tris buffer and MgCl2Mixing and dissolving with NaCl; then adding reduced nicotinamide adenine dinucleotide, glucose-6-phosphate, carbitol and dimethyl sulfoxide to obtain a recombinant glucose-6-phosphate dehydrogenase solution;
(C2) preparation of 17-hydroxysteroid derivative solution: dissolving a 17-hydroxysteroid derivative shown in formula (I) in claim 1 in dimethylformamide, cooling to-18 ℃, adding tributylamine and isobutyl chloroformate, stirring at low temperature and mixing uniformly to obtain a 17-hydroxysteroid derivative solution;
(C3) obtaining the 17-hydroxysteroid enzyme-labeled conjugate: and (4) dropwise adding the 17-hydroxysteroid derivative solution obtained in the step (C2) into the recombinant glucose-6-phosphate dehydrogenase solution obtained in the step (C1), stirring overnight, and purifying by using a gel chromatography column to obtain the 17-hydroxysteroid enzyme-labeled conjugate.
7. A preparation method of a 17-hydroxysteroid detection reagent is characterized by comprising the following steps:
(D1) preparation of homogeneous enzyme substrate: dissolving oxidized nicotinamide adenine dinucleotide and glucose-6-phosphate in a Tris buffer solution according to the ratio of the final concentration to the final concentration of 1:1 to prepare a homogeneous enzyme substrate;
(D2) preparation of reagent A: uniformly mixing the homogeneous enzyme substrate obtained in step (D1) and the anti-17-hydroxysteroid specific antibody as described in claim 1 to obtain reagent a, wherein the volume ratio of the anti-17-hydroxysteroid specific antibody to the homogeneous enzyme substrate in reagent a is 1: 50-5000;
(D3) preparation of reagent B: dissolving the 17-hydroxysteroid enzyme-labeled conjugate in Tris buffer solution to obtain a reagent B, wherein the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer solution in the reagent B is 1: 50-5000.
8. The process according to claim 7, wherein the volume ratio of the anti-17-hydroxysteroid specific antibody to the homogeneous enzyme substrate in the reagent A is 1: 500;
the volume ratio of the 17-hydroxysteroid enzyme-labeled conjugate to the Tris buffer solution in the reagent B is 1: 750.
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