CN110003300A - A kind of derivative of 17OHS, detection reagent and preparation method - Google Patents
A kind of derivative of 17OHS, detection reagent and preparation method Download PDFInfo
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- CN110003300A CN110003300A CN201910325085.3A CN201910325085A CN110003300A CN 110003300 A CN110003300 A CN 110003300A CN 201910325085 A CN201910325085 A CN 201910325085A CN 110003300 A CN110003300 A CN 110003300A
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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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- G01N33/531—Production of immunochemical test materials
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- 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
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Abstract
The invention discloses a kind of derivative of 17OHS, detection reagent and preparation methods, are related to technical field of biological.The 17OHS immunogene prepared using the 17OHS derivative, immunogenicity is high, and obtained antibody specificity is strong, potency is high；17OHS enzyme mark conjugate is connected to the recombination glucose-6-phosphate dehydrogenase (G6PD) being genetically engineered in the homogeneous enzyme immunoassay detection reagent being prepared using the derivative, significantly improve detection sensitivity, can effectively detectable concentration down to 80nmol/L sample, and high specificity, with 92 kinds of common chaff interferent no cross reactions；It may be implemented in high-throughput, the rapid detection on automatic clinical chemistry analyzer to 17OHS content, testing result is stablized, and accuracy is high, and detection method is simple, it is easy to accomplish and promote the use of.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of derivative of 17OHS, detection reagent and
Preparation method.
Background technique
17OHS (17-hydorxycorticosteroids, 17-OHCS), shown in structural formula such as formula (IV):
17OHS belongs to glucocorticoid, is the hormone as secreted by adrenal cortex and its metabolism
Product mainly has pure and mild tetrahydro cortex element of cortisol, cortin, tetrahydro cortex etc., often excretes with urine.Therefore, urine
The content of middle 17OHS can reflect the case where adrenocortical secretion cortisol, can auxiliary diagnosis correlation endocrine disease
Disease.The reduction of 17OHS content is common in adrenocortical insufficiency, adenohypophysis hypofunction, bilateral adrenal glands
After resection, malnutrition, cirrhosis, tuberculosis and certain chronic wasting diseases etc.；17OHS content increases then
It is high to be common in adrenocortical hyperfunction (tumour or hyperplasia cause), ectopic ACTH syndrome, obesity, thyroid function
Into disease and pancreatitis etc..
The method of 17OHS is colorimetric method in most common detection urine.This method principle is: in acid
Property under the conditions of, 17OHS and phenylhydrazine hydrochloride act on, and generate yellow phenylhydrazine derivant, this colour developing result subtracts sulfuric acid and its
Background color caused by its non-17OHS colour developing object, processing colorimetric same as titer can seek its content.This method
Detecting step includes: acquisition by inspection urine；Extract 17OHS sample；Prepare colorimetric estimation sample；Carry out chromogenic reaction；
Colorimetric reads absorbance, calculates 17OHS content according to absorbance.This method need to use chloroform-n-butanol mixing
Liquid is stripped, which has anesthetic effect and irritation, and hydrogen chloride can be oxidized under the action of light and has severe toxicity
Phosgene brings harm to operator and environment；On the other hand, the result stability detected using this method is poor, and operation is multiple
Miscellaneous, time-consuming, is not suitable for carrying out a large amount of clinical applications.
With the continuous development of Measurement for Biotechnique, the detection method of 17OHS is gradually improved.In the prior art
In, Chinese patent CN103335969B discloses a kind of colorimetric estimation method of 17-hydorxycorticosteroids, and this method is free of trichlorine
The toxic reagents such as methane, chloroform, safe operation process is reliable, without using anhydrous slufuric acid ammonium and anhydrous sodium sulfate, saves reagent；
By the way that traditional spare test solution of extracting is modified to after depigmentation directly enormously simplify using supernatant by inspection urine preparation
The step of at spare test solution, measuring method is simple to operation.Chinese patent CN105092831A discloses a kind of 17- hydroxyl cortex class
Sterol immunologic function test reagent and preparation method thereof, the reagent include anti-17OHS specific antibody and indicator, most
Low detectable concentration is 1umol/L.Currently, it is less with existing detection reagent in the market, and there are still certain defects, such as:
Stability is poor, sensitivity is low, is unsuitable for a large amount of samples while detection etc..
Therefore, in view of the deficiencies in the prior art, research and develop a kind of quality and reach clinical requirement, high sensitivity, stablize
Property is good, result is accurate, and the reagent that can be applied to high-throughput, the rapid detection of automatic clinical chemistry analyzer has become domestic ectosome
The hot spot of outer diagnostic reagent industry.
Summary of the invention
The purpose of the present invention is to provide a kind of derivative of 17OHS, detection reagent and preparation methods.It overcomes
Defect of the existing technology, the homogeneous enzyme immunoassay detection reagent with high sensitivity, stability is good, and high specificity is, it can be achieved that big
Batch sample automatically quickly detects.
One aspect of the present invention provides a kind of 17OHS derivative, the structure of the 17OHS derivative
Shown in formula such as formula (I):
The present invention also provides a kind of preparation method of above-mentioned 17OHS derivative, the preparation method packets
Include following steps:
(1) synthesis of compound 2: compound 1 and triethylamine are dissolved in methylene chloride, and dihydrofuran -2,5- is added
Diketone, stirring, thin-layered chromatography is shown after the reaction was completed, with purified water quenching reaction, salt acid for adjusting pH, methylene chloride extraction 3
It is secondary, merge organic layer, with purified water and brine, vacuum drying concentration obtains white solid, i.e. compound 2；
(2) synthesis of compound 3: the compound 2 that step (1) obtains is dissolved in pyridine, is added Pd (OH)2, in room
Mild H2Under the conditions of be stirred overnight, thin-layered chromatography display starting reaction raw materials all disappear after, filter, be concentrated in vacuo, purifying
Obtain white solid, i.e. compound 3；
(3) synthesis of 17OHS derivative: the compound 3 that step (2) obtains is dissolved in anhydrous tetrahydro furan,
At -78 DEG C, triisobutyl potassium borohydride, stirring, after thin-layered chromatography shows fully reacting, with saturation NH is added4Cl
Aqueous solution quenching reaction, ethyl acetate extract 3 times, merge organic layer, and with purified water and brine, vacuum drying concentration is pure
Change obtains white solid, i.e. 17OHS derivative；
The synthesis path of the 17OHS derivative is as follows:
Specifically, the preparation method of above-mentioned 17OHS derivative, comprising the following steps:
(1) 5g compound 1 and 2.1g triethylamine (TEA) synthesis of compound 2: are dissolved in methylene chloride (DCM) together
In (50mL), it is then added at one time 1.8g dihydrofuran -2,5- diketone at room temperature, is stirred at room temperature 2 hours, thin layer color
Spectrometry (TLC) is shown after the reaction was completed, with 300mL purified water quenching reaction, pH=3 is adjusted with the hydrochloric acid of 1N, after reaction
Mixture is extracted with 300mL DCM, is repeated 3 times, and is merged organic layer, is washed with purified water (200mL) and brine (200mL)
It washs, is dried and is concentrated in a vacuum, obtain white solid, i.e. compound 2；
(2) synthesis of compound 3: the compound 2 that step (1) obtains is dissolved in pyridine (120mL), 4g is then added
Pd(OH)2, in room temperature and H2Under the conditions of be stirred overnight, thin-layered chromatography display starting reaction raw materials all disappear after, by what is obtained
Solution is filtered, then filtrate is concentrated in a vacuum, and obtained crude product obtains white solid, i.e., through silica gel purification
Compound 3；
(3) compound 3 that step (2) obtains the synthesis of 17OHS derivative: is dissolved in anhydrous tetrahydro furan
(THF) in (50mL), at -78 DEG C, triisobutyl potassium borohydride (15mL, 1M) is added dropwise, is stirred 1 hour at -78 DEG C,
After thin-layered chromatography display reaction has been fully completed, with saturation NH4Then Cl aqueous solution quenching reaction uses ethyl acetate (EA)
(200mL) is extracted, and is repeated 3 times, and is merged organic layer, is washed with purified water (200mL) and brine (200mL), in a vacuum
It dries and concentrates, obtained crude product is purified by silica gel, obtains white solid, i.e. 17OHS derivative.
The present invention also provides a kind of 17OHS detection reagent, the 17OHS detection reagent includes examination
Agent A and reagent B；
The reagent A includes anti-17OHS specific antibody and homogeneous zymolyte；
The reagent B includes 17OHS enzyme mark conjugate and Tris buffer；
The anti-17OHS specific antibody is by generating after 17OHS immunogen immune experimental animal
Complete antibody molecule, or to retain and the antibody fragment or antibody derivatives of 17OHS specific binding capacity；
The experimental animal is mammal；Preferably, the experimental animal be sheep, goat, mouse, cavy,
One of rabbit or horse；It is highly preferred that the experimental animal is sheep.
The 17OHS immunogene is by above-mentioned 17OHS derivative or above-mentioned preparation method system
The compound that standby obtained 17OHS derivative is connect with carrier, shown in structural formula such as formula (II):
The carrier is the protein or polypeptide with immunogenicity；
Preferably, the carrier is one of thyroglobulin, haemocyanin or hemocyanin；It is highly preferred that
The carrier is thyroglobulin；
The homogeneous zymolyte is by G-6-P, the nicotinamide adenine dinucleotide of oxidation state and Tris
What buffer obtained；
The 17OHS enzyme mark conjugate is by above-mentioned 17OHS derivative or above-mentioned preparation method
The 17OHS derivative being prepared is formed by connecting with recombination glucose-6-phosphate dehydrogenase (G6PD) (rG6PDH), structural formula
As shown in formula (III):
Specifically, the amino acid sequence of glucose-6-phosphate dehydrogenase (G6PD) (rG6PDH) is recombinated as shown in SEQ ID NO:1.
Preferably, the 17OHS immunogene preparation method the following steps are included:
(A1) preparation of carrier solution: carrier is dissolved in phosphate buffer, carrier solution is obtained；
(A2) preparation of 17OHS derivative solution: by above-mentioned 17OHS derivative, dimethyl formyl
Amine, ethyl alcohol, kaliumphosphate buffer, 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, N- hydroxy thiosuccinimide are mixed
It closes, stirring and dissolving reacts 90min, obtains 17OHS derivative solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to
It in the carrier solution that step (A1) obtains, and is stirred overnight at 4 DEG C, through dialysis purification, obtains 17OHS immunogene.
Specifically, the 17OHS immunogene preparation method the following steps are included:
(A1) preparation of carrier solution: being dissolved in 0.2M for carrier protein, in the phosphate buffer of pH=8.5, carrier egg
White final concentration of 4mg/mL, obtains carrier solution；
(A2) preparation of 17OHS derivative solution: by 100-300mg above-mentioned 17OHS derivative,
1.75-5.25mL dimethylformamide, 1.75-5.25mL ethyl alcohol, 3.5-10.5mL, the potassium phosphate buffering of 10mM, pH=5.0
Liquid, 100-300mg 1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide, 25-75mg N- hydroxy thiosuccinimide are mixed
It closes, stirring and dissolving reacts 30-60min, obtains 17OHS derivative solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to
It in the carrier solution that step (A1) obtains, and is stirred overnight at 4 DEG C, through dialysis purification, obtains 17OHS immunogene.
Preferably, the anti-17OHS specific antibody preparation method the following steps are included:
(B1) above-mentioned 17OHS immunogene is diluted with PBS, obtains antigenic solution, it is then that antigen is molten
Liquid is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
(B2) after 5-6 weeks, then with identical antigenic solution above-mentioned experimental animal is carried out with equivalent incomplete Freund's adjuvant
Injection, it is primary every injection in 2 weeks later, amount to injection 5-8 times；
(B3) blood is taken to the experimental animal of step (B2), isolated and purified, obtain anti-17OHS specific antibody.
Specifically, the anti-17OHS specific antibody preparation method the following steps are included:
(B1) above-mentioned 17OHS immunogene is diluted to 0.1-3.0mg/mL with PBS, obtains antigenic solution, so
Antigenic solution is mixed with equivalent Freund's complete adjuvant afterwards, experimental animal is injected；
(B2) after 5-6 weeks, then with identical antigenic solution above-mentioned experimental animal is carried out with equivalent incomplete Freund's adjuvant
Injection, it is primary every injection in 2 weeks later, amount to injection 5-8 times；
(B3) blood is taken to the experimental animal of step (B2), isolated and purified, obtain anti-17OHS specific antibody.
Preferably, the 17OHS enzyme mark conjugate preparation method the following steps are included:
(C1) it recombinates the preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: glucose-6-phosphate dehydrogenase (G6PD), Tris will be recombinated
Buffer, MgCl2With NaCl mixed dissolution；Add the nicotinamide adenine dinucleotide of reduction-state, G-6-P,
Carbitol and dimethyl sulfoxide obtain recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) above-mentioned 17OHS derivative the preparation of 17OHS derivative solution: is dissolved in dimethyl
In formamide, -18 DEG C are cooled to, tri-n-butylamine is added and isobutyl chlorocarbonate, low temperature stir and evenly mix, obtains 17OHS and spreads out
Biological solution；
(C3) obtain 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) is obtained by
It is added dropwise in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution that step (C1) obtains, 2-8 DEG C is stirred overnight, gel chromatography column
Purifying, obtains 17OHS enzyme mark conjugate.
Specifically, the 17OHS enzyme mark conjugate preparation method the following steps are included:
(C1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution is recombinated: by the recombination G-6-P of 7.5-22.5mg
Dehydrogenase, 6-18mL Tris buffer, 8mg MgCl2With 100mg NaCl mixed dissolution；Add 112.5-337.5mg also
Nicotinamide adenine dinucleotide, 67.5-202.5mg G-6-P, 0.375-1.125mL carbitol and the 1- of ortho states
3mL dimethyl sulfoxide obtains recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) preparation of 17OHS derivative solution: the above-mentioned 17OHS derivative of 5-15mg is dissolved in
In 300-900 μ l dimethylformamide, -18 DEG C are cooled to, 1.5-4.5 μ l tri-n-butylamine is added and 0.75-2.25 μ l chloro-carbonic acid is different
Butyl ester, low temperature stir and evenly mix, and obtain 17OHS derivative solution；
(C3) obtain 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) is obtained by
It is added dropwise in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution that step (C1) obtains, 2-8 DEG C is stirred overnight, gel chromatography column
Purifying, obtains 17OHS enzyme mark conjugate.
Another aspect of the present invention provides a kind of preparation method of above-mentioned 17OHS detection reagent, including following
Step:
(D1) preparation of homogeneous zymolyte: the nicotinamide adenine dinucleotide of oxidation state and G-6-P are pressed
It is dissolved in Tris buffer according to final concentration than the ratio for 1:1, homogeneous zymolyte is made；
(D2) preparation of reagent A: the homogeneous zymolyte and above-mentioned anti-17OHS that above-mentioned steps (D1) are obtained are special
Heterogenetic antibody is uniformly mixed, and obtains reagent A, anti-17OHS specific antibody and homogeneous zymolyte in the reagent A
Volume ratio is 1:50-5000；
(D3) preparation of reagent B: above-mentioned 17OHS enzyme mark conjugate is dissolved in Tris buffer, is tried
The volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:50-5000 in agent B, the reagent B.
Preferably, the volume ratio of anti-17OHS specific antibody and homogeneous zymolyte is 1 in the reagent A:
500；The volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:750 in the reagent B.
The invention has the benefit that
The present invention provides a kind of derivative of 17OHS, detection reagent and preparation methods.It is prepared by the derivative
Obtained antibody specificity is strong, potency is high, and 17- hydroxyl class is solid in the homogeneous enzyme immunoassay detection reagent being prepared using the derivative
Alcoholase mark conjugate is connected to the recombination glucose-6-phosphate dehydrogenase (G6PD) (rG6PDH) being genetically engineered, and significantly improves
Detection sensitivity, can effective sample of the detectable concentration down to 80nmol/L；It may be implemented on automatic clinical chemistry analyzer to 17-
High-throughput, the rapid detection of hydroxysteroid content, the stability of detection and accuracy are high, high specificity, with common 92 kinds
Chaff interferent no cross reaction, detection efficiency significantly improve, and detection method is simple, it is easy to accomplish and promote the use of.
Detailed description of the invention
Fig. 1 is the ELISA examination criteria curve of 17OHS in the embodiment of the present invention 5；
Fig. 2 is the homogeneous enzyme immunoassay standard curve of 17OHS in the embodiment of the present invention 12.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention, it should be noted that not
Under the premise of conflicting, new implementation can be formed between various embodiments described below or between each technical characteristic in any combination
Example.
The synthesis of embodiment 1:17- hydroxysteroid derivative
The synthesis path of 17OHS derivative is as follows:
The preparation method of 17OHS derivative, the specific steps are as follows:
(1) 5g compound 1 and 2.1g triethylamine (TEA) synthesis of compound 2: are dissolved in methylene chloride (DCM) together
In (50mL), it is then added at one time dihydrofuran -2,5- diketone 1.8g at room temperature, is stirred at room temperature 2 hours, thin layer color
Spectrometry (TLC) is shown after the reaction was completed, with 300mL purified water quenching reaction, pH=3 is adjusted with the hydrochloric acid of 1N, after reaction
Mixture is extracted with 300mL DCM, is repeated 3 times, and is merged organic layer, is washed with purified water (200mL) and brine (200mL)
It washs, is dried and is concentrated in a vacuum, obtain white solid 6g, i.e. compound 2, yield: 94%；
(2) synthesis of compound 3: the compound 2 (6g) that step (1) obtains is dissolved in pyridine (120mL), then plus
Enter Pd (OH)2(4g), in room temperature and H2Under the conditions of be stirred overnight, thin-layered chromatography display starting reaction raw materials all disappear after, will
Obtained solution is filtered, then filtrate is concentrated in a vacuum, and it is solid to obtain white through silica gel purification for obtained crude product
Body 3.5g, i.e. compound 3, yield: 58.3%；
(3) compound 3 (3.5g) that step (2) obtains the synthesis of 17OHS derivative: is dissolved in anhydrous tetrahydro
In furans (THF) (50mL), triisobutyl potassium borohydride (15mL, 1M) is added dropwise at -78 DEG C, it is small that 1 is stirred at -78 DEG C
When, after thin-layered chromatography display reaction has been fully completed, with saturation NH4Cl aqueous solution quenching reaction, then uses ethyl acetate
(EA) (200mL) is extracted, and is repeated 3 times, and is merged organic layer, is washed with purified water (200ml) and brine (200ml), true
Aerial to dry and concentrate, obtained crude product is purified by silica gel, obtains white solid 1.1g, yield: 31.4%.
Using Bruker Avance III plus 400MHz and VARIAN MERCURY plus 300M to step (3)
Obtained compound as white solid carries out NMR spectrum scanning and identifies its chemical structure using TMS as internal standard.As a result
It is as follows:
1H NMR(CD3OD,400MHz):δ0.54-0.55(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).
Shown in its structural formula such as formula (I):
The result shows that the white solid is 17OHS derivative.
The preparation of embodiment 2:17- hydroxysteroid immunogene and anti-17OHS specific antibody
The preparation step of 17OHS immunogene is as follows:
(A1) preparation of hemocyanin solution: hemocyanin 100mg is dissolved in 25mL, the phosphoric acid of 0.2M, pH=8.5 are slow
In fliud flushing, hemocyanin solution is obtained；
(A2) preparation of 17OHS derivative solution: the 17OHS that 100mg embodiment 1 is obtained is derivative
Object, 1.75mL dimethylformamide, 1.75mL ethyl alcohol, 3.5mL, the kaliumphosphate buffer of 10mM, pH=5.0,100mg 1- second
Base -3- (- 3- dimethylaminopropyl) carbodiimide, the mixing of 25mg N- hydroxy thiosuccinimide, stirring and dissolving react 90min,
Obtain 17OHS derivative solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to
It in the hemocyanin solution that step (A1) obtains, and is stirred overnight at 4 DEG C, through dialysis purification, it is immune to obtain 17OHS
It is former.
The preparation step of anti-17OHS specific antibody is as follows:
(B1) above-mentioned 17OHS immunogene is diluted to 0.1mg/mL with PBS, obtains antigenic solution, then will
Antigenic solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal goat；
(B2) after 5 weeks, then with identical antigenic solution and equivalent incomplete Freund's adjuvant to above-mentioned experimental animal goat into
Row injection, it is primary every injection in 2 weeks later, amount to injection 5 times；
(B3) blood is taken to the experimental animal goat of step (B2), isolates and purifies to obtain the anti-17- hydroxyl class that potency is 1:30000
Sterol specific antibody.
The preparation of embodiment 3:17- hydroxysteroid immunogene and anti-17OHS specific antibody
The preparation step of 17OHS immunogene is as follows:
(A1) thyroglobulin 200mg the preparation of thyroglobulin solution: is dissolved in 50mL, 0.2M, pH=8.5
Phosphate buffer in, obtain thyroglobulin solution；
(A2) preparation of 17OHS derivative solution: the 17OHS that 200mg embodiment 1 is obtained is derivative
Object, 3.5mL dimethylformamide, 3.5mL ethyl alcohol, 7.0mL, kaliumphosphate buffer, the 200mg 1- ethyl-of 10mM, pH=5.0
3- (- 3- dimethylaminopropyl) carbodiimide, the mixing of 50mg N- hydroxy thiosuccinimide, stirring and dissolving are reacted 90min, are obtained
To 17OHS derivative solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to
It in the thyroglobulin solution that step (A1) obtains, and is stirred overnight at 4 DEG C, through dialysis purification, obtains 17OHS
Immunogene.
The preparation step of anti-17OHS specific antibody is as follows:
(B1) above-mentioned 17OHS immunogene is diluted to 1.0mg/mL with PBS, obtains antigenic solution, then will
Antigenic solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal sheep；
(B2) after 5 weeks, then with identical antigenic solution and equivalent incomplete Freund's adjuvant to above-mentioned experimental animal sheep into
Row injection, it is primary every injection in 2 weeks later, amount to injection 6 times；
(B3) blood is taken to the experimental animal sheep of step (B2), isolates and purifies to obtain the anti-17- hydroxyl class that potency is 1:50000
Sterol specific antibody.
The preparation of embodiment 4:17- hydroxysteroid immunogene and anti-17OHS specific antibody
The preparation step of 17OHS immunogene is as follows:
(A1) preparation of haemocyanin solution: haemocyanin 300mg is dissolved in 75mL, the phosphoric acid of 0.2M, pH=8.5 are slow
In fliud flushing, haemocyanin solution is obtained；
(A2) preparation of 17OHS derivative solution: the 17OHS that 300mg embodiment 1 is obtained is derivative
Object, 5.25mL dimethylformamide, 5.25mL ethyl alcohol, 10.5mL, the kaliumphosphate buffer of 10mM, pH=5.0,300mg 1- second
Base -3- (- 3- dimethylaminopropyl) carbodiimide, the mixing of 75mg N- hydroxy thiosuccinimide, stirring and dissolving react 90min,
Obtain 17OHS derivative solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to
It in the haemocyanin solution that step (A1) obtains, and is stirred overnight at 4 DEG C, through dialysis purification, it is immune to obtain 17OHS
It is former.
The preparation step of anti-17OHS specific antibody is as follows:
(B1) above-mentioned 17OHS immunogene is diluted to 3.0mg/mL with PBS, obtains antigenic solution, then will
Antigenic solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal mouse；
(B2) after 6 weeks, then with identical antigenic solution and equivalent incomplete Freund's adjuvant to above-mentioned experimental animal mouse into
Row injection, it is primary every injection in 2 weeks later, amount to injection 8 times；
(B3) blood is taken to the experimental animal mouse of step (B2), isolates and purifies to obtain the anti-17- hydroxyl class that potency is 1:40000
Sterol specific antibody.
The ELISA of embodiment 5:17- hydroxysteroid is examined
(1) the ELISA examination criteria curve of 17OHS is established
The preparation of standard items: 17OHS powder (being purchased from Sigma company) is dissolved in methanol solution, is prepared into
The storing liquid of 1000 μm of ol/L.Storing liquid is successively diluted to 800.0 with ELISA buffer, 400.0,200.0,100.0,
50.0, the standard solution of 0.0nmol/L.Wherein, ELISA buffer contains 50.0mM Tris, 145mM NaCl and mass fraction
For 0.25% bovine serum albumin(BSA) (BSA).
It draws standard curve: anti-17OHS specific antibody prepared by embodiment 3 being diluted to 1 with PBS:
8000 final concentration solution, 100 holes μ l/ are coated on 96 hole elisa plates, 4 DEG C of placement 12-24h, will be coated with PBS above-mentioned anti-
96 hole elisa plates of body wash 3 times, and the BSA solution that the mass fraction in 200 holes μ l/ is 0.5% is added, and 8- is placed in 4 DEG C of closings
16h is washed 3 times with PBS, and the standard items in 20 holes μ l/ are added, add the horseradish peroxidase of 100 hole μ l/ working concentrations
(HRP) -17OHS conjugate is incubated for 30min at room temperature, and PBS board-washing 5 times, then 100 μ l tetramethyls connection is added in every hole
Aniline (TMB) substrate is incubated at room temperature 30min, then 100 μ l terminate liquids (i.e. 2M sulfuric acid) are added in every hole.It is surveyed in the case where wavelength is 450nm
Determine light absorption value, makes standard curve, as a result as shown in Figure 1.
(2) in sample to be tested 17OHS content detection
The preparation of sample to be tested: 17OHS powder (being purchased from Sigma company) is dissolved in methanol solution and is made 1000
The storing liquid of μm ol/L, and this storing liquid is diluted in blank diaper, until final concentration is respectively 0.00,80.00,320.00,
800.00nmol/L, forms blank, the urine specimen of basic, normal, high concentration, which is strong without 17OHS
Health human urine.
Detect sample to be tested: using the method for inspection in above-mentioned steps (1), by the urine sample of blank, basic, normal, high concentration
This replaces standard items, and each sample carries out 3 multiple holes measurements, and detection blank, the urine specimen of basic, normal, high concentration are at 450nm
Light absorption value.Standard curve according to figure 1 calculates the content of 17OHS in each sample, according to 17- in sample
The actual content of hydroxysteroid calculates the rate of recovery, and the rate of recovery=detectable concentration/sample concentration × 100%, the results are shown in Table 1.
The ELISA testing result of table 1:17- hydroxysteroid
| Urine sample | Blank | It is low | In | It is high |
| 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 |
| The rate of recovery (%) | - | 100.9 | 99.3 | 101.0 |
As seen from the results in Table 1: using in 17OHS ELISA detection reagent of the present invention measurement various concentration sample
The 17OHS rate of recovery is all higher, reaches the 99% < rate of recovery≤101%, illustrates anti-17OHS provided by the invention
Specific antibody can be used for the detection of 17OHS in sample, and result precision is high, and stability is good.
Consolidate in addition, the present invention has also carried out 17- hydroxyl class to the anti-17OHS specific antibody of other embodiments preparation
The ELISA of alcohol is detected, and the 17OHS rate of recovery is higher, can reach 95% < rate of recovery < 105%.
The preparation of embodiment 6:17- hydroxysteroid enzyme mark conjugate
(C1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution is recombinated: by the recombination G-6-P dehydrogenation of 7.5mg
Enzyme (amino acid sequence is as shown in SEQ ID NO:1), 6mL Tris buffer, 8mg MgCl2With 100mg NaCl mixed dissolution；
Add the nicotinamide adenine dinucleotide of 112.5mg reduction-state, 67.5mg G-6-P, 0.375mL carbitol and
1mL dimethyl sulfoxide obtains recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) preparation of 17OHS derivative solution: the 17OHS derivative that 5mg embodiment 1 is obtained is molten
Solution cools to -18 DEG C in 300 μ l dimethylformamides, is added 1.5 μ l tri-n-butylamines and 0.75 μ l isobutyl chlorocarbonate, -2~-
8 DEG C of stirring 30min mix, obtain 17OHS derivative solution；
(C3) obtain 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) is obtained by
It is added dropwise in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution that step (C1) obtains, 2-8 DEG C is stirred overnight, gel chromatography column
Purifying, obtains 17OHS enzyme mark conjugate.
The preparation of embodiment 7:17- hydroxysteroid enzyme mark conjugate
(C1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution is recombinated: by the recombination glucose-6-phosphate dehydrogenase (G6PD) of 15mg
(amino acid sequence is as shown in SEQ ID NO:1), 12mL Tris buffer, 8mg MgCl2With 100mg NaCl mixed dissolution；
Add nicotinamide adenine dinucleotide, 135mg G-6-P, 0.75mL carbitol and the 2mL of 225mg reduction-state
Dimethyl sulfoxide obtains recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) preparation of 17OHS derivative solution: the 17OHS derivative that 10mg embodiment 1 is obtained
It is dissolved in 600 μ l dimethylformamides, cools to -18 DEG C, be added 3 μ l tri-n-butylamines and 1.5 μ l isobutyl chlorocarbonates, -2~-8
DEG C stirring 30min, mix, obtain 17OHS derivative solution.
(C3) obtain 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) is obtained by
It is added dropwise in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution that step (C1) obtains, 2-8 DEG C is stirred overnight, gel chromatography column
Purifying, obtains 17OHS enzyme mark conjugate.
The preparation of embodiment 8:17- hydroxysteroid enzyme mark conjugate
(C1) preparation of glucose-6-phosphate dehydrogenase (G6PD) solution is recombinated: by the recombination G-6-P dehydrogenation of 22.5mg
Enzyme (amino acid sequence is as shown in SEQ ID NO:1), 18mL Tris buffer, 8mg MgCl2It is mixed with 100mg NaCl molten
Solution；Adding the nicotinamide adenine dinucleotide of 337.5mg reduction-state, 202.5mg G-6-P, 1.125mL card must
Pure and mild 3mL dimethyl sulfoxide obtains recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) preparation of 17OHS derivative solution: the 17OHS derivative that 15mg embodiment 1 is obtained
It is dissolved in 900 μ l dimethylformamides, cools to -18 DEG C, be added 4.5 μ l tri-n-butylamines and 2.25 μ l isobutyl chlorocarbonates, -2
~-8 DEG C of stirring 30min mix, obtain 17OHS derivative solution.
(C3) obtain 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) is obtained by
It is added dropwise in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution that step (C1) obtains, 2-8 DEG C is stirred overnight, gel chromatography column
Purifying, obtains 17OHS enzyme mark conjugate.
The preparation of embodiment 9:17- hydroxysteroid detection reagent
Specifically includes the following steps:
(D1) preparation of homogeneous zymolyte: by the nicotinamide adenine dinucleotide (NAD) of 12.5g oxidation state and the Portugal 5.2g
The Tris buffer solution of grape sugar -6- phosphoric acid (G-6-P) 3L80mM, pH=8.5, are made homogeneous zymolyte, wherein oxidation state
Nicotinamide adenine dinucleotide and G-6-P final concentration ratio be 1:1；
(D2) preparation of reagent A: anti-17- hydroxyl made from the homogeneous zymolyte and embodiment 3 that above-mentioned steps (D1) are obtained
Steroids specific antibody is uniformly mixed, and obtains reagent A, anti-17OHS specific antibody and homogeneous zymolyte in reagent A
Volume ratio be 1:50；
(D3) the 17OHS enzyme mark conjugate that embodiment 7 obtains the preparation of reagent B: is dissolved in 100mM, pH=7.8
Tris buffer in, obtain reagent B, the volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1 in reagent B:
50。
The preparation of embodiment 10:17- hydroxysteroid detection reagent
The present embodiment and the difference of embodiment 9 be, anti-17OHS specific antibody and homogeneous enzyme bottom in reagent A
The volume ratio of object is 1:500；The volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:750 in reagent B.
The preparation of embodiment 11:17- hydroxysteroid detection reagent
The present embodiment and the difference of embodiment 9 be, anti-17OHS specific antibody and homogeneous enzyme bottom in reagent A
The volume ratio of object is 1:5000；The volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:5000 in reagent B.
Embodiment 12:17- hydroxysteroid homogeneous enzyme immunoassay is examined
(1) 17OHS homogeneous enzyme immunoassay test stone curve is established
The preparation of standard items: preparation method is identical as the ELISA examination criteria product preparation method in embodiment 5.
It draws standard curve: stepping auspicious BS-480 automatic clinical chemistry analyzer response parameter according to the setting of table 2.17- hydroxyl used
Steroids homogeneous enzyme immunoassay testing reagent is the detection reagent that embodiment 10 is prepared.First reagent adding A, adds standard items,
It is eventually adding reagent B.After reagent B is added, the OD of different time points is measured340Light absorption value, when calculating various criterion product concentration
Reaction rate draws reaction normal curve, as shown in Figure 2.
Table 2: auspicious BS-480 automatic clinical chemistry analyzer response parameter is stepped
| Project name | 17OHS |
| Reagent A | 200μl |
| Reagent B | 50μl |
| Sample size | 12μl |
| Analysis method | End-point method |
| Dominant wavelength | 340nm |
| Secondary wavelength | 412nm |
| Reaction time | 10 minutes |
| Incubation time | 5 minutes |
| The Direction of Reaction | Rise |
| As a result | nmol/L |
| As a result precision | 0.01 |
| Calibrating method | Logistic-Log 5P |
| Standard concentration | 0.00,50.00,100.00,200.00,400.00,800.00nmol/L |
(2) sample to be tested detects:
Sample to be tested is that 17OHS standard items are dissolved in human urine, until concentration is respectively 80.00,320.00,
800.00nmol/L.The basic, normal, high concentration Quality Control sample of replication 10 times, standard curve according to Fig.2, calculate each
The content and the rate of recovery of 17OHS in sample, the rate of recovery=detectable concentration/sample concentration × 100%, as a result such as 3 institute of table
Show.
Table 3: sample measurement and precision and rate of recovery assessment
| Urine sample | It is low | In | It is high |
| 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 |
| Rate of recovery % | 101.1 | 100.9 | 100.7 |
Testing result: the accuracy of homogeneous enzyme immunoassay detection reagent measurement of the invention is high, and the rate of recovery reaches 95%-
105%, precision is high, and CV is below 3%.
13. drug of embodiment and hormone interference test
It chooses 62 kinds of Common drugs and 30 kinds of common hormones and hormone metabolism object carries out Interference Detection, adjust chaff interferent to be measured
Concentration is detected to 0.10 μm of ol/L using the 17OHS homogeneous enzyme immunoassay detection method of embodiment 12.
Reagent A haptoreaction prepared by chaff interferent to be measured and embodiment 10, adds reagent B；Detect the suction at 340nm
Light value obtains the concentration of respective substance according to standard curve as shown in Figure 2.62 kinds of Common drugs and 30 kinds of common hormones and swash
Plain metabolin title and measurement result are referring specifically to table 4.
Table 4: common interference object homogeneous enzyme immunoassay inspection result
Measurement result is shown: above-mentioned 62 kinds of Common drugs and 30 kinds of common hormones and hormone metabolism object are equivalent to 17- hydroxyl class
The concentration of sterol is respectively less than 0.10 μm of ol/L.It can be seen that 17OHS homogeneous enzyme immunoassay provided by the invention examines examination
Agent, high specificity, with 92 kinds of common chaff interferent no cross reactions.
Embodiment described above is only to absolutely prove preferred embodiment that is of the invention and being lifted, protection model of the invention
It encloses without being limited thereto.Those skilled in the art's made equivalent substitute or transformation on the basis of the present invention, in the present invention
Protection scope within.Protection scope of the present invention is subject to claims.
Sequence table
<110>Suzhou Bo Yuan medical science and technology Co., Ltd
<120>a kind of derivative of 17OHS, detection reagent and preparation method
<130> 2019
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 455
<212> PRT
<213>artificial synthesized (Artificial)
<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 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 (10)
1. a kind of 17OHS derivative, which is characterized in that the structural formula such as formula (I) of the 17OHS derivative
It is shown:
2. a kind of preparation method of 17OHS derivative described in claim 1, which is characterized in that the preparation side
Method the following steps are included:
(1) synthesis of compound 2: compound 1 and triethylamine are dissolved in methylene chloride, and dihydrofuran -2,5- diketone is added,
Stirring, thin-layered chromatography is shown after the reaction was completed, and with purified water quenching reaction, salt acid for adjusting pH, methylene chloride is extracted 3 times, is closed
And organic layer, with purified water and brine, vacuum drying concentration obtains white solid, i.e. compound 2；
(2) synthesis of compound 3: the compound 2 that step (1) obtains is dissolved in pyridine, is added Pd (OH)2, in room temperature and H2
Under the conditions of be stirred overnight, thin-layered chromatography display starting reaction raw materials all disappear after, filter, vacuum concentration, purifying obtain it is white
Color solid, i.e. compound 3；
(3) synthesis of 17OHS derivative: the compound 3 that step (2) obtains is dissolved in anhydrous tetrahydro furan, -78
At DEG C, triisobutyl potassium borohydride, stirring, after thin-layered chromatography shows fully reacting, with saturation NH is added4Cl aqueous solution
Quenching reaction, ethyl acetate extract 3 times, merge organic layer, and with purified water and brine, vacuum drying concentration, purifying is obtained
White solid, i.e. 17OHS derivative；
The synthesis path of the 17OHS derivative is as follows:
3. a kind of 17OHS detection reagent, which is characterized in that the 17OHS detection reagent include reagent A and
Reagent B；
The reagent A includes anti-17OHS specific antibody and homogeneous zymolyte；
The reagent B includes 17OHS enzyme mark conjugate and Tris buffer；
The anti-17OHS specific antibody is complete by generating after 17OHS immunogen immune experimental animal
Whole antibody molecule, or to retain antibody fragment or antibody derivatives with 17OHS specific binding capacity；
The experimental animal is mammal；
The 17OHS immunogene is by 17OHS derivative described in claim 1 or claim 2 institute
The compound that the 17OHS derivative that the preparation method stated is prepared is connect with carrier, structural formula such as formula
(II) shown in:
The carrier is the protein or polypeptide with immunogenicity；
The homogeneous zymolyte is buffered by G-6-P, the nicotinamide adenine dinucleotide of oxidation state and Tris
What liquid was prepared；
The 17OHS enzyme mark conjugate is by 17OHS derivative described in claim 1 or claim
The 17OHS derivative that 2 preparation methods are prepared is formed by connecting with recombination glucose-6-phosphate dehydrogenase (G6PD),
Shown in structural formula such as formula (III):
4. 17OHS detection reagent according to claim 3, which is characterized in that the carrier is thyroid gland ball
One of albumen, haemocyanin or hemocyanin；
The experimental animal is one of sheep, goat, mouse, cavy, rabbit or horse.
5. 17OHS detection reagent according to claim 4, which is characterized in that the carrier is thyroid gland ball egg
It is white；The experimental animal is sheep.
6. 17OHS detection reagent according to claim 3, which is characterized in that the 17OHS is immune
Former preparation method the following steps are included:
(A1) preparation of carrier solution: carrier is dissolved in phosphate buffer, carrier solution is obtained；
(A2) preparation of 17OHS derivative solution: by 17OHS derivative described in claim 1 or right
It is required that the 17OHS derivative that is prepared of 2 preparation methods and dimethylformamide, ethyl alcohol, kaliumphosphate buffer,
1- ethyl -3- (- 3- dimethylaminopropyl) carbodiimide and the mixing of N- hydroxy thiosuccinimide, stirring and dissolving obtain 17- hydroxyl
Steroid derivatives solution；
(A3) it obtains 17OHS immunogene: the 17OHS derivative solution that step (A2) obtains is added to step
(A1) it in the carrier solution obtained, is stirred overnight, through dialysis purification, obtains 17OHS immunogene.
7. 17OHS detection reagent according to claim 3, which is characterized in that the anti-17OHS is special
The preparation method of heterogenetic antibody the following steps are included:
(B1) 17OHS immunogene described in claim 3 is diluted with PBS, antigenic solution is obtained, then by antigen
Solution is mixed with equivalent Freund's complete adjuvant, is injected to experimental animal；
(B2) after 5-6 weeks, then with identical antigenic solution above-mentioned experimental animal is injected with equivalent incomplete Freund's adjuvant,
It is primary every injection in 2 weeks later, amount to injection 5-8 times；
(B3) blood is taken to the experimental animal of step (B2), isolated and purified, obtain anti-17OHS specific antibody.
8. 17OHS detection reagent according to claim 3, which is characterized in that the 17OHS enzyme mark
The preparation method of conjugate the following steps are included:
(C1) it recombinates the preparation of glucose-6-phosphate dehydrogenase (G6PD) solution: recombination glucose-6-phosphate dehydrogenase (G6PD), Tris is buffered
Liquid, MgCl2With NaCl mixed dissolution；Adding the nicotinamide adenine dinucleotide of reduction-state, G-6-P, card must
Pure and mild dimethyl sulfoxide obtains recombination glucose-6-phosphate dehydrogenase (G6PD) solution；
(C2) preparation of 17OHS derivative solution: by 17OHS derivative described in claim 1 or right
It is required that the 17OHS derivative that 2 preparation methods are prepared is dissolved in dimethylformamide, -18 DEG C are cooled to,
Tri-n-butylamine is added and isobutyl chlorocarbonate, low temperature stir and evenly mix, obtains 17OHS derivative solution；
(C3) it obtains 17OHS enzyme mark conjugate: the 17OHS derivative solution that step (C2) obtains is added dropwise
Enter in the recombination glucose-6-phosphate dehydrogenase (G6PD) solution obtained to step (C1), be stirred overnight, gel chromatography column purification obtains
17OHS enzyme mark conjugate.
9. a kind of preparation method of 17OHS detection reagent, which is characterized in that the preparation method comprises the following steps:
(D1) preparation of homogeneous zymolyte: by the nicotinamide adenine dinucleotide of oxidation state and G-6-P according to end
Concentration is dissolved in Tris buffer than the ratio for 1:1, and homogeneous zymolyte is made；
(D2) preparation of reagent A: anti-17- hydroxyl class described in the homogeneous zymolyte and claim 3 that step (D1) is obtained is solid
Alcohol specific antibody is uniformly mixed, and obtains reagent A, anti-17OHS specific antibody and homogeneous enzyme bottom in the reagent A
The volume ratio of object is 1:50-5000；
(D3) preparation of reagent B: the mark conjugate of 17OHS enzyme described in claim 3 is dissolved in Tris buffer,
Reagent B is obtained, the volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:50-5000 in the reagent B.
10. 17OHS detection reagent according to claim 9, which is characterized in that anti-17- hydroxyl in the reagent A
The volume ratio of steroids specific antibody and homogeneous zymolyte is 1:500；
The volume ratio of 17OHS enzyme mark conjugate and Tris buffer is 1:750 in the reagent B.
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