CN107973836B - Aldosterone derivative, preparation method thereof and aldosterone homogeneous enzyme immunoassay reagent - Google Patents
Aldosterone derivative, preparation method thereof and aldosterone homogeneous enzyme immunoassay reagent Download PDFInfo
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- CN107973836B CN107973836B CN201710977334.8A CN201710977334A CN107973836B CN 107973836 B CN107973836 B CN 107973836B CN 201710977334 A CN201710977334 A CN 201710977334A CN 107973836 B CN107973836 B CN 107973836B
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- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J43/00—Normal steroids having a nitrogen-containing hetero ring spiro-condensed or not condensed with the cyclopenta(a)hydrophenanthrene skeleton
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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
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
Abstract
The invention discloses an aldosterone derivative and a preparation method thereof, an aldosterone homogeneous enzyme immunoassay reagent and a preparation method and a detection method thereof. The aldosterone derivative has a structure shown in a formula (I), the aldosterone immunogen and the antibody thereof with strong immunogenicity are prepared from the aldosterone derivative, and the aldosterone homogeneous enzyme immunoassay reagent prepared by the antibody can realize high-throughput and rapid detection of aldosterone on a full-automatic biochemical analyzer.
Description
Technical Field
The invention relates to the technical field of aldosterone detection, in particular to an aldosterone derivative and a preparation method thereof, an aldosterone homogeneous enzyme immunoassay reagent and a preparation method and a detection method thereof.
Background
The structure of Aldosterone (Aldosterone) is shown as formula (II):
aldosterone is a mineralocorticoid hormone synthesized and secreted by adrenal cortex zona cells, mainly acts on renal distal convoluted tubule and renal cortex collecting duct to increase reabsorption of sodium ions and promote excretion of potassium ions, and also acts on medullary collecting duct to promote excretion of hydrogen ions and acidify urine. The secretion of aldosterone is achieved by the renin-angiotensin-aldosterone system. When the extracellular fluid volume is reduced, the extracellular fluid volume is stimulated to secrete renin by the glomerular side cells, a renin-angiotensin-aldosterone system is activated, aldosterone secretion is increased, and sodium reabsorption of the kidney is increased, so that water reabsorption is increased and the extracellular fluid volume is increased; conversely, when the extracellular fluid volume increases, the secretion of aldosterone decreases, the reabsorption of sodium and water by the kidneys decreases, and the extracellular fluid volume decreases, by the reverse mechanism described above. Blood sodium is reduced and blood potassium is increased to stimulate adrenal cortex and increase aldosterone secretion.
Increased aldosterone secretion suggests a possible association with the following clinical conditions: 1. primary aldosteronism caused by adrenal cortical hyperplasia and the like; 2. high secondary hyperaldosteronism caused by hypothalamic-pituitary dysfunction, ectopic corticotropin secretion, etc.; 3. non-specific increase of aldosterone caused by liver cirrhosis, renal hypertension, multiple renal cysts, etc. The pathological reduction of aldosterone is seen primarily in primary adrenocortical insufficiency, also known as Addison's disease.
The renin-angiotensin-aldosterone system is a series of circulating hormones synthesized and secreted by multiple organs of the body, and plays the roles of regulating blood pressure, water and electrolyte balance and maintaining the environment in the body, and the action mechanism of the renin-angiotensin-aldosterone system comprises the regulation of systemic blood volume and the control of peripheral resistance. A large number of clinical trial researches show that the renin-angiotensin-aldosterone system coordinates and acts on a plurality of systems such as cardiovascular system, urinary system, nervous system and the like, and plays an important physiological role in maintaining the balance of blood pressure and blood volume of the organism. When the organism has hypertension, respiratory system and urinary system diseases, a series of abnormal changes occur on the average of the renin-angiotensin-aldosterone level, and the renin-angiotensin-aldosterone level can be accurately measured, thereby having very important significance for diagnosis and treatment of corresponding diseases.
At present, the clinical determination of aldosterone content in serum, plasma or urine has many methods, mainly including enzyme-linked immunosorbent assay, chemiluminescence method, magnetic particle chemiluminescence method, enhanced chemiluminescence immunoassay method, high performance liquid chromatography and the like, but the existing methods have certain limitations, such as complex operation, poor stability, need of special instruments, high detection cost and the like, and are not suitable for determination of large-scale clinical samples. At present, aldosterone detection reagents with good stability, high sensitivity and strong specificity, especially automatic detection reagents with good quality, are lacked in the market. Therefore, the development and production quality meets the clinical requirements, the practicability is strong, the cost performance is high, and the aldosterone determination kit which can be applied to the full-automatic biochemical analyzer becomes a hotspot in the in vitro diagnostic reagent industry at home and abroad.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an aldosterone derivative, an aldosterone immunogen with strong immunogenicity and an antibody thereof are prepared from the aldosterone derivative, and an aldosterone homogeneous enzyme immunoassay reagent prepared from the antibody can realize high-throughput and rapid detection of aldosterone on a full-automatic biochemical analyzer.
The second purpose of the invention is to provide a preparation method of aldosterone derivative.
The invention also aims to provide an aldosterone homogeneous enzyme immunoassay reagent prepared from the aldosterone derivatives.
The fourth purpose of the invention is to provide a preparation method of the aldosterone homogeneous enzyme immunoassay reagent,
the fifth purpose of the invention is to provide a detection method of aldosterone homogeneous enzyme immunoassay reagent.
One of the purposes of the invention is realized by adopting the following technical scheme:
an aldosterone derivative has a structure as shown in formula (I),
the second purpose of the invention is realized by adopting the following technical scheme:
a preparation method of an aldosterone derivative, the aldosterone derivative has a structure as shown in formula (I), the preparation method comprises the following steps:
(1) reacting the compound 4 with methanesulfonyl halide and lithium chloride to generate a compound 5;
(2) compound 5 and
reacting to generate a compound 6;
(3) reacting the compound 6 with Boc anhydride to generate a compound 7;
(4) reacting the compound 7 with HCl to generate a compound 8;
(5) compound 8 with SOCl2Reacting to generate a compound 9;
(6) compound 9 with NaBH4Reacting to generate a compound 10;
(7) compound 10 with maleimide, PPh3And DIAD to generate the aldosterone derivative shown in the formula (I).
The third purpose of the invention is realized by adopting the following technical scheme:
an aldosterone homogeneous enzyme immunoassay reagent comprising: an anti-aldosterone-specific antibody, an indicator reagent for detecting an anti-aldosterone-specific antibody-aldosterone complex; the anti-aldosterone specific antibody is obtained by immunizing experimental animals with aldosterone immunogen, the aldosterone immunogen is formed by connecting an aldosterone derivative shown in formula (I) and a carrier, and the carrier is protein with immunogenicity; the indicator reagent is selected from an enzyme reagent, a radioisotope reagent, a fluorescent reagent, or a chemiluminescent reagent.
Optionally, the indicator reagent is selected from enzymatic reagents, including: enzyme-labeled conjugates and enzyme substrates; the enzyme-labeled conjugate comprises a glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate; the substrate of the enzyme is glucose-6-phosphate; the glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate is formed by reacting glucose-6-phosphate dehydrogenase with an aldosterone derivative shown in a formula (I).
The fourth purpose of the invention is realized by adopting the following technical scheme:
the preparation method of the aldosterone homogeneous enzyme immunoassay reagent comprises the following steps:
(1) synthesis of aldosterone immunogen: linking an aldosterone derivative represented by formula (I) with a protein carrier having immunogenicity to produce an aldosterone immunogen;
(2) preparation of anti-aldosterone specific antibody: immunizing experimental animals by using the aldosterone immunogen to obtain an anti-aldosterone specific antibody from the experimental animals;
(3) preparation of glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate: preparing a glucose-6-phosphate dehydrogenase solution, activating the aldosterone derivative shown in the formula (I), and connecting the glucose-6-phosphate dehydrogenase with the aldosterone derivative shown in the formula (I) to obtain a glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate;
(4) preparing an aldosterone homogeneous enzyme immunoassay reagent:
preparation of reagent A: the antibody is formed by mixing the anti-aldosterone specific antibody and a homogeneous enzyme substrate;
preparation of reagent B: the enzyme-labeled conjugate of glucose-6-phosphate dehydrogenase-hapten and Tris buffer solution are mixed to prepare the enzyme-labeled conjugate.
Optionally, in the step (1), the protein carrier is BSA, and the step of synthesizing the aldosterone immunogen is as follows:
a. weighing 2.72g of monopotassium phosphate, 4.26g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution A;
b. weighing 3mg of BSA, and dissolving in 3mL of the buffer solution A at room temperature to prepare a BSA solution;
c. weighing 3mg of aldosterone derivative shown in formula (I), and dissolving in 300ul of the buffer solution A to obtain aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the BSA solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution A to obtain a solution, namely an aldosterone immunogen solution, and adding NaN with the mass fraction of 0.1 percent into the aldosterone immunogen solution3And storing at-20 ℃.
Optionally, the step (2) includes:
a. diluting the aldosterone immunogen in the step (1) to 1.0mg/ml by PBS to obtain an antigen solution, mixing 1.0ml of the antigen solution with Freund's complete adjuvant, and injecting the experimental animal;
after 2-3 weeks, mixing 1.0ml of the same antigen solution with Freund's incomplete adjuvant, injecting the experimental animal once, and then injecting every 3-4 weeks for 4-6 times in total;
c. and (3) taking blood from the immunized experimental animal, separating and purifying to obtain the anti-aldosterone specific antibody with the titer of 1:30000-1: 50000.
Optionally, the step (3) includes:
a. weighing 1.09g of potassium dihydrogen phosphate, 1.70g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution B;
b. weighing 3mg of glucose-6-phosphate dehydrogenase, and dissolving the glucose-6-phosphate dehydrogenase in 3mL of the buffer solution B at room temperature to prepare a glucose-6-phosphate dehydrogenase solution;
c. weighing 3mg of aldosterone derivative shown in the formula (I), and dissolving the aldosterone derivative in 300ul of the buffer solution B to prepare aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution B to obtain a solution, namely a glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate solution, and adding BSA with the mass fraction of 0.5% and NaN with the mass fraction of 0.1% into the glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate solution3And storing at 2-8 deg.C.
Optionally, the step (4) includes:
preparation of reagent A: dissolving 5.0g of nicotinamide adenine dinucleotide in an oxidized state and 2.5g of glucose-6-phosphate in 1L of 55mM Tris buffer (pH 8.0) to prepare a homogeneous enzyme substrate; adding the prepared anti-aldosterone specific antibody into the homogeneous enzyme substrate, wherein the volume ratio of the antibody to the homogeneous enzyme substrate is 1: 100-1: 10000;
preparation of reagent B: adding the prepared glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate into 120mM Tris buffer solution with the pH value of 8.2, wherein the volume ratio of the conjugate to the Tris buffer solution is 1: 100-1: 10000.
The fifth purpose of the invention is realized by adopting the following technical scheme:
a detection method of an aldosterone homogeneous phase enzyme immunoassay reagent is provided, wherein the aldosterone homogeneous phase enzyme immunoassay reagent is the aldosterone homogeneous phase enzyme immunoassay reagent; the detection method comprises the following steps:
(1) contacting a sample to be detected with an anti-aldosterone specific antibody;
(2) according to the combination condition of aldosterone and an anti-aldosterone specific antibody in a sample to be detected, judging the content of aldosterone in the sample by using an indicating reagent;
the sample to be detected is a biological sample, and the biological sample is serum, plasma, urine, saliva or milk.
Compared with the prior art, the invention has the beneficial effects that:
in the invention, the aldosterone immunogen prepared from the aldosterone derivative shown in the formula (1) has strong specificity and high immunogenicity, and the prepared anti-aldosterone specific antibody has strong specificity and high titer, and does not have any cross reaction with 92 common interferents; the homogeneous enzyme immunoassay reagent containing the anti-aldosterone specific antibody can conveniently, quickly and accurately determine the aldosterone content in a sample, can simultaneously determine a plurality of samples on a full-automatic biochemical analyzer, realizes high-flux quick determination of aldosterone, has high accuracy and specificity, greatly improves the accuracy and the detection efficiency compared with the prior art, simultaneously realizes the full automation of the detection process, has low requirements on detection personnel, is easy to realize, popularize and use, and can effectively meet the increasing clinical detection requirements in China.
Drawings
FIG. 1 is a reaction curve of ELISA detection of aldosterone according to the present invention.
FIG. 2 is a graph showing the reaction curve of the homogeneous enzyme immunoassay of aldosterone according to the present invention.
FIG. 3 is a diagram showing the correlation between the aldosterone homogeneous enzyme immunoassay method and high performance liquid chromatography method 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.
The invention provides an aldosterone derivative, aldosterone immunogen with strong immunogenicity and an antibody thereof are prepared from the aldosterone derivative, and aldosterone homogeneous enzyme immunoassay reagent prepared by using the antibody can realize high-throughput and rapid detection of aldosterone on a full-automatic biochemical analyzer. The aldosterone derivative has a structure shown in a formula (I),
the first embodiment is as follows: the synthetic route for preparing the aldosterone derivative shown in the formula (I) is as follows:
specifically, the preparation method of the aldosterone derivative shown as the formula (I) comprises the following steps:
(1) reacting the compound 4 with methanesulfonyl halide (MsCl) and lithium chloride (LiCl) to generate a compound 5;
(2) compound 5 and
reacting to generate a compound 6;
(3) compound 6 with Boc anhydride (Boc)2O) to generate a compound 7;
(4) reacting the compound 7 with HCl to generate a compound 8;
(5) compound 8 with SOCl2Reacting to generate a compound 9;
(6) compound 9 with NaBH4Reacting to generate a compound 10;
(7) compound 10 with maleimide, PPh3And DIAD to generate the aldosterone derivative shown in the formula (I).
Wherein the content of the first and second substances,
the synthetic route of (2) is as follows:
(1) reacting the compound 1 with benzyl chloroformate (CbzCl) to generate a compound 2;
(2) reacting the compound 2 with tert-butyl alcohol to generate a compound 3;
(3) compound 3 reacts with palladium carbon (Pd/C) to form
(Amine1)。
The specific synthetic steps are as follows:
synthesis of Compound 2:
55 g of a catalyst (Cbz-Cl, benzyl chloroformate) was added in one portion to a 2N equivalent (2mol/L) sodium hydroxide solution containing 30 g of Compound 1 (4-aminobutyric acid) dissolved therein at 10 ℃. The mixture was stirred at room temperature overnight. During this time, the progress of the reaction was monitored by TLC until the reaction was complete. The reacted mixture was extracted with ethyl acetate (3X 300mL, 3 times 300mL each). The aqueous layer was adjusted to pH 3 with 1N equivalent hydrochloric acid and extracted with dichloromethane (3 × 300 mL). The combined organic layers were washed with clean water (200mL), brine (200mL), dried under vacuum, and concentrated to give Compound 2 as a white solid (33g, 48.8% yield).
Synthesis of Compound 3
87 g of Dicyclohexylcarbodiimide (DCC) was added in one portion to a solution of 33g of Compound 2 dissolved in 500mL of dichloromethane, 17 g of 4-Dimethylaminopyridine (DMAP) and 41mL of t-butanol at 10 ℃. The mixture was stirred at room temperature overnight. During this time, the progress of the reaction was monitored by TLC until the reaction was complete. The solution after the reaction was filtered to collect the filtrate. The filtrate was diluted with water and the pH was adjusted to 3 with 1N normal hydrochloric acid. The resulting mixture was extracted with dichloromethane (3X 500mL), and the organic layers were combined and washed with water (300mL), brine (300 mL). After washing, vacuum drying and concentrating, a crude product is obtained. The crude product was purified by column chromatography on silica gel to give compound 3(26g, 62.4% yield) as a white solid.
Synthesis of Compound Amine1
To a solution of 26g of compound 3 dissolved in 250mL of methanol was added 8g of palladium on carbon (Pd/C) to obtain a mixture. The mixture was stirred overnight at 50 ℃ under hydrogen as a blanket gas. TLC controlled the progress of the reaction until the starting reaction disappeared and the resulting solution was filtered. And vacuum drying and concentrating the filtrate to obtain a crude product. The crude product was purified by column chromatography on silica gel to give Amine1 as a white solid (14g, 99.2% yield).
Synthesis of Compound 5
A mixture was prepared by adding 4.1 g of methanesulfonyl chloride (MsCl) dropwise to a solution of 10 g of Compound 4, 0.34 g of 4-dimethylaminopyridine and 5.6 g of Triethylamine (TEA) dissolved in 200mL of dichloromethane at 0 ℃. The mixture was stirred at room temperature for 1 hour. TLC controlled the progress of the reaction until the reaction was complete, then cooled the reaction with 50mL of methanol, dried in vacuo, and concentrated to a yellow oil. The yellow oil was dissolved in 150mL of Dimethylformamide (DMF), and 1.53 g of lithium chloride (LiCl) was added to the mixture, and after stirring at 60 ℃ for 2 hours, the reaction was added to water, and the precipitate was collected by filtration to give compound 5(8.5 g, yield 80.9%) as a white solid.
Synthesis of Compound 6
1g of Compound 5 and 0.68 g of N, N-Diisopropylethylamine (DIEA) were dissolved in 15mL of dimethyl sulfoxide to obtain a solution, and then 0.84 g of Amine1 was added to the solution at room temperature. The mixture was stirred at 40 ℃ for 3 hours. The progress of the reaction was monitored by liquid chromatography-mass spectrometry (LC/MS) until the reaction was complete. The reacted solution was quenched with 100mL of water, then extracted with ethyl acetate (3X 300mL), and the organic layers were combined. The organic phase was washed with water (100mL), brine (100 mL). After washing, vacuum drying and concentrating, a crude product is obtained. The crude product was purified by silica gel column chromatography to give compound 6(0.41 g, 30.8% yield) as a yellow solid.
Synthesis of Compound 7
6.3 g of Compound 6, 2.54 g of Triethylamine (TEA) were dissolved in 60mL of dichloromethane to obtain a solution. 5.5g of Boc anhydride (Boc) was added at 10 deg.C2O) was added dropwise to the solution described above. The mixture was stirred at room temperature for 1 hour. TLC controls the reaction progress until the reaction is complete. And drying and concentrating the reacted solution in vacuum to obtain a crude product. The crude product was purified by column chromatography on silica gel to give compound 7(6 g, 79.4% yield) as a yellow solid.
Synthesis of Compound 8
3.5 g of Compound 7 were dissolved in a 6N-strength hydrochloric acid/dioxane (80mL) solution and stirred at room temperature for 5 hours. The progress of the reaction was monitored by liquid chromatography-mass spectrometry (LC/MS) until disappearance of the starting reactant. The reacted mixture was diluted with 200mL of methyl tert-butyl ether (MTBE) and the precipitate was filtered to give compound 8(2.3 g, 82.1% yield) as a white solid.
Synthesis of Compound 9
5.5g of Compound 8 are weighed out, dissolved in 50mL of MeOH and 4.0g (34.1mmol) of SOCl are added dropwise at 0 deg.C2Then, the reaction mixture was stirred at 70 ℃ overnight, the solvent was evaporated under reduced pressure, and the residue was removedThe retentate was passed through 200mL of DCM and 200mL of NaHCO3Separating with saturated aqueous solution, washing the organic phase with brine, and passing through Na2SO4Drying and concentration gave 5.1g of compound 9 as a white solid in 93% yield.
Synthesis of Compound 10
5.1g of Compound 9 are weighed out, dissolved in 100mL of MeOH and 1.3g (33.3mmol) of NaBH are added in portions at 0 ℃4Then, the reaction mixture was stirred at room temperature overnight, the reaction mixture was concentrated, and the residue obtained after the concentration was purified by silica gel dry column (DCM: MeOH ═ 10: 1) to obtain 4.6g of compound 10 as a colorless oil in 90% yield.
Synthesis of aldosterone derivative represented by formula (1)
2.3g of Compound 10 are weighed out, dissolved in 40mL of THF, and 1.2g (12.6mmol) of maleimide and 3.8g (14.56mmol) of PPh are added under nitrogen protection at 0 deg.C3And 2.9g (14.56mmol) of DIAD, and the reaction mixture was stirred at 70 ℃ overnight, the reaction mixture was concentrated, and the residue obtained after concentration was purified by silica gel dry column (DCM: MeOH ═ 50: 1) to obtain 800mg of final compound as a tan solid in 35% yield. The final compound is the aldosterone derivative shown in formula (I).
Example two: synthesis of aldosterone immunogens
The aldosterone immunogen is formed by connecting Bovine Serum Albumin (BSA) and aldosterone derivatives shown in a formula (I), and the synthesis method of the immunogen comprises the following specific steps:
a. weighing 2.72g of monopotassium phosphate, 4.26g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution A;
b. weighing 3mg of BSA, and dissolving in 3mL of the buffer solution A at room temperature to prepare a BSA solution;
c. weighing 3mg of aldosterone derivative shown in formula (I), and dissolving in 300ul of the buffer solution A to prepare aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the BSA solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution A to obtain a solution, namely an aldosterone immunogen solution, and adding NaN with the mass fraction of 0.1 percent into the aldosterone immunogen solution3And storing at-20 ℃. 0.1% NaN3The adding amount is the mass percentage of the finally obtained immunogen solution, and the specific adding amount is determined according to the specific mass of the immunogen solution obtained after dialysis.
Example three: preparation of anti-aldosterone specific antibodies
Inoculating the aldosterone immunogen prepared in the second embodiment to an experimental animal, taking an experimental animal rabbit as an example, taking antiserum after enhancing immunity, and specifically comprising the following steps:
1. the aldosterone immunogen synthesized by the method is diluted to 1.0mg/ml by PBS to obtain an antigen solution, and then the antigen solution of 1.0ml is mixed with Freund's complete adjuvant to inject the experimental animal rabbit.
After 2.3 weeks, 1.0ml of the same antigen solution was mixed with Freund's incomplete adjuvant and injected once into the above experimental animal rabbits every 4 weeks for a total of 6 injections.
3. And (3) taking blood from the immunized experimental animal rabbit, separating and purifying to obtain the anti-aldosterone specific antibody with the titer of 1:30000-1: 50000.
Example four: aldosterone ELISA assay
1. Establishment of aldosterone ELISA detection standard curve
(1) Preparation of standards
Aldosterone powder (purchased from Sigma) was dissolved in methanol solution to prepare a stock solution of 1 mg/ml. The stock solutions were diluted sequentially with ELISA buffer to 160.00pg/mL, 80.00pg/mL, 40.00pg/mL, 20.00pg/mL, 10.00pg/mL, and 0.00pg/mL of standard solutions. The ELISA buffer contained 50.0mM Tris, 145mM NaCl and 0.25% BSA.
(2) Preparation of a Standard Curve by ELISA test method Using aldosterone
The anti-aldosterone antibody prepared in example three was diluted with PBS to 1: coating 100 mu L/well of 9000 final concentration solution on a 96-well enzyme-linked plate, and standing at 4 ℃ for 12-24 h; after washing the 96-well ELISA plate coated with the anti-aldosterone antibody for 3 times with PBS, 200. mu.L/well of 0.5% BSA solution was added, and the plate was blocked at 4 ℃ and left standing for 8-16 h. Then, the cells were washed 3 times with PBS, and 20. mu.L/well of a standard was added. Then adding HRP-aldosterone conjugate with the working concentration of 100 mu L/hole; incubating for 30min at room temperature, and washing the plate for 5 times by PBS; then 100. mu.L of TMB substrate was added to each well and incubated for 30min at room temperature. Then 100. mu.L of stop solution (2M sulfuric acid) was added to each well. Absorbance at 450nm was measured. The standard curve was prepared by calibration according to the absorbance value of 450nm corresponding to each standard, and the result is shown in FIG. 1.
2. Detection of aldosterone content in sample to be detected
(1) Making a sample to be tested
The preparation method comprises the following steps: aldosterone powder (purchased from Sigma) was dissolved in methanol solution to make a stock solution of 1 μ g/mL, and this stock solution was diluted in blank plasma to final concentrations of 0.00, 1.00, 20.00, 100.00pg/mL, respectively, to form blank, low, medium, and high concentration plasma samples. The blank plasma is healthy human plasma without aldosterone.
(2) Test method
And (3) testing the absorbance values of the blank, low, medium and high-concentration plasma samples at 450nm by using the ELISA test method for the aldosterone, wherein the blank, low, medium and high-concentration plasma samples replace the standard substance.
(3) Test results
The results of calculating the aldosterone content in each sample and performing 3 replicate well assays on each sample, in comparison to the standard curve of the aldosterone ELISA test shown in figure 1, are shown in table 1, based on the actual aldosterone content in said sample.
TABLE 1 ELISA test for aldosterone recovery
From the results in table 1, it can be seen that: the aldosterone recovery rates of samples with different concentrations measured by the aldosterone ELISA detection reagent are higher and are all higher than 95%, which shows that the anti-aldosterone specific antibody can be used for detecting aldosterone in a sample and the result accuracy is high.
Example five: preparation of glucose-6-phosphate dehydrogenase-hapten conjugates
a. Weighing 1.09g of potassium dihydrogen phosphate, 1.70g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution B;
b. weighing 3mg of glucose-6-phosphate dehydrogenase, and dissolving the glucose-6-phosphate dehydrogenase in 3mL of the buffer solution B at room temperature to prepare a glucose-6-phosphate dehydrogenase solution;
c. weighing 3mg of aldosterone derivative shown in formula (I), and dissolving in 300ul of the buffer solution B to prepare aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution B to obtain a solution, namely a glucose-6-phosphate dehydrogenase-hapten conjugate solution, and adding BSA with the mass fraction of 0.5% and NaN with the mass fraction of 0.1% into the glucose-6-phosphate dehydrogenase-hapten conjugate solution3And storing at 2-8 deg.C. 0.5% BSA and 0.1% NaN3The adding amount is the mass percentage of the finally obtained conjugate solution, and the specific adding amount is determined according to the specific mass of the conjugate solution obtained after dialysis.
Example six: preparation of aldosterone homogeneous enzyme immunoassay reagent
Before the aldosterone homogeneous enzyme immunoassay reagent is used, in order to avoid the enzyme-labeled conjugate in the indicator reagent from reacting with the enzyme substrate, the enzyme-labeled conjugate and the enzyme substrate are separately placed and not mixed, so that the enzyme substrate and the anti-aldosterone specific antibody are mixed together. That is, the aldosterone homogeneous enzyme immunoassay reagent comprises two separately disposed reagents, specifically as follows:
preparation of reagent A: dissolving 5.0g of nicotinamide adenine dinucleotide in an oxidized state and 2.5g of glucose-6-phosphate in 1L of 55mM Tris buffer (pH 8.0) to prepare a homogeneous enzyme substrate; adding the prepared anti-aldosterone specific antibody into the homogeneous enzyme substrate, wherein the volume ratio of the antibody to the homogeneous enzyme substrate is 1: 100-1: 10000;
the volume ratio of the anti-aldosterone specific antibody to the homogeneous enzyme substrate in this example was specifically 1: 700.
Preparation of reagent B: adding the prepared glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate into 120mM Tris buffer solution with the pH value of 8.2, wherein the volume ratio of the conjugate to the Tris buffer solution is 1: 100-1: 10000.
The volume ratio of the aldosterone enzyme-labeled conjugate to the Tris buffer solution in the embodiment is specifically 1: 2800.
Example seven: aldosterone homogeneous enzyme immunoassay and results
1. Obtaining a standard curve:
(1) the reaction parameters of the michi BS480 full-automatic biochemical analyzer were set (see table 2).
(2) The operation steps are as follows: adding the reagent A, adding the standard substance and finally adding the reagent B. After the reagent B is added, OD340 light absorption values at different time points are measured, the reaction rates of different standard substance concentrations are calculated, the volume ratio of the reagent A to the reagent B needs to be continuously adjusted in the actual operation process, the light measurement point is adjusted at the same time, and finally, a relatively ideal reaction standard curve chart is obtained, as shown in FIG. 2.
TABLE 2 Merrill BS480 fully automated Biochemical Analyzer reaction parameters
2. Sample detection: the standard curve obtained by the homogeneous enzyme immunoassay reagent of the invention is used for repeatedly measuring low, medium and high concentration quality control samples for 10 times, wherein the quality control samples are as follows: the aldosterone standard was dissolved in human plasma to concentrations of 2.00, 25.00, 90.00pg/ml, respectively. The data obtained and the data analysis are shown in Table 3.
TABLE 3 sample determination and precision and recovery evaluation
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 eight: drug and hormone intervention test
Selecting 62 common medicines and 30 common hormones and hormone metabolites for interference detection, adjusting the concentration to 1.00ng/ml, and adopting the homogeneous enzyme immunoassay method of the seventh embodiment to carry out determination:
1. the interferent to be detected is contacted and reacted with the reagent A prepared in the sixth embodiment, and then the reagent B is added;
2. and detecting the OD340 light absorption value of the mixed solution, and obtaining the concentration of the corresponding substance according to the standard curve of the seventh embodiment.
The names and the measurement results of 62 common drugs and 30 common hormones and hormone metabolites are shown in Table 4.
TABLE 4 common interferent assay results
The measurement results show that: the concentrations of the 62 common medicaments, the 30 common hormones and hormone metabolites equivalent to aldosterone are less than 1.00 pg/ml. It can be seen that the antibodies of the invention are specific antibodies against aldosterone and do not cross-react with common interferents.
Example nine: correlation analysis
Correlation analysis was performed on 100 clinical specimens using HPLC and the homogeneous enzyme immunoreagent of the present invention, and the measured data are shown in Table 5.
TABLE 5 clinical sample measurements
Plotting the above data, see FIG. 3, the resulting linear equation is: y 1.0015x +0.1489, correlation coefficient R2The detection reagent of the invention is 0.9994, which shows that the detection reagent has high accuracy in detecting aldosterone clinical specimens.
Various other modifications and changes may be made by those skilled in the art based on the above-described technical solutions and concepts, and all such modifications and changes should fall within the scope of the claims of the present invention.
Claims (7)
1. An aldosterone derivative is characterized in that the aldosterone derivative has a structure as shown in formula (I),
2. a preparation method of an aldosterone derivative, wherein the aldosterone derivative has a structure as shown in formula (I), the preparation method comprises the following steps:
(1) reacting the compound 4 with methanesulfonyl halide and lithium chloride to generate a compound 5;
(2) compound 5 and
reacting to generate a compound 6;
(3) reacting the compound 6 with Boc anhydride to generate a compound 7;
(4) reacting the compound 7 with HCl to generate a compound 8;
(5) compound 8 with SOCl2Reacting to generate a compound 9;
(6) compound 9 with NaBH4Reacting to generate a compound 10;
(7) compound 10 with maleimide, PPh3And DIAD to generate the aldosterone derivative shown in the formula (I).
3. An aldosterone immunogen comprising the aldosterone derivative of claim 1 linked to a carrier, said carrier being BSA.
4. The preparation method of the aldosterone homogeneous enzyme immunoassay reagent is characterized by comprising the following steps:
(1) synthesis of aldosterone immunogen: linking the aldosterone derivative of claim 1 to a protein carrier that is immunogenic to produce an aldosterone immunogen;
(2) preparation of anti-aldosterone specific antibody: immunizing experimental animals by using the aldosterone immunogen to obtain an anti-aldosterone specific antibody from the experimental animals;
(3) preparation of glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate:
a. weighing 1.09g of potassium dihydrogen phosphate, 1.70g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution B;
b. weighing 3mg of glucose-6-phosphate dehydrogenase, and dissolving the glucose-6-phosphate dehydrogenase in 3mL of the buffer solution B at room temperature to prepare a glucose-6-phosphate dehydrogenase solution;
c. weighing 3mg of the aldosterone derivative of claim 1 and dissolving the aldosterone derivative in 300ul of the buffer solution B to form an aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the glucose-6-phosphate dehydrogenase solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution B to obtain a solution, namely a glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate solution, and adding BSA with the mass fraction of 0.5% and NaN with the mass fraction of 0.1% into the glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate solution3Storing at 2-8 deg.C;
(4) preparing an aldosterone homogeneous enzyme immunoassay reagent:
preparation of reagent A: the antibody is formed by mixing the anti-aldosterone specific antibody and a homogeneous enzyme substrate;
preparation of reagent B: the enzyme-labeled conjugate of glucose-6-phosphate dehydrogenase-hapten and Tris buffer solution are mixed to prepare the enzyme-labeled conjugate.
5. The method according to claim 4, wherein the step (1) of synthesizing the aldosterone immunogen comprises the steps of:
a. weighing 2.72g of monopotassium phosphate, 4.26g of disodium hydrogen phosphate, 8.5g of sodium chloride and 0.95g of magnesium chloride, dissolving the components in 1L of deionized water, and adjusting the pH value to 7.4 to prepare a buffer solution A;
b. weighing 3mg of BSA, and dissolving in 3mL of the buffer solution A at room temperature to prepare a BSA solution;
c. weighing 3mg of the aldosterone derivative of claim 1 and dissolving the aldosterone derivative in 300ul of the buffer solution A to form an aldosterone derivative solution;
d. when the aldosterone derivative solution just becomes clear, the aldosterone derivative solution is added dropwise to the BSA solution, and then the mixed solution is stirred at 2-8 ℃ for 1 hour;
e. dialyzing the reacted mixed solution with the buffer solution A to obtain a solution, namely an aldosterone immunogen solution, and adding NaN with the mass fraction of 0.1 percent into the aldosterone immunogen solution3And storing at-20 ℃.
6. The method according to claim 4, wherein the step (2) comprises:
a. diluting the aldosterone immunogen in the step (1) to 1.0mg/ml by PBS to obtain an antigen solution, mixing 1.0ml of the antigen solution with Freund's complete adjuvant, and injecting the experimental animal;
after 2-3 weeks, mixing 1.0ml of the same antigen solution with Freund's incomplete adjuvant, injecting the experimental animal once, and then injecting every 3-4 weeks for 4-6 times in total;
c. and (3) taking blood from the immunized experimental animal, separating and purifying to obtain the anti-aldosterone specific antibody with the titer of 1:30000-1: 50000.
7. The method according to claim 4, wherein the step (4) comprises:
preparation of reagent A: dissolving 5.0g of nicotinamide adenine dinucleotide in an oxidized state and 2.5g of glucose-6-phosphate in 1L of 55mM Tris buffer (pH 8.0) to prepare a homogeneous enzyme substrate; adding the prepared anti-aldosterone specific antibody into the homogeneous enzyme substrate, wherein the volume ratio of the antibody to the homogeneous enzyme substrate is 1: 100-1: 10000;
preparation of reagent B: adding the prepared glucose-6-phosphate dehydrogenase-hapten enzyme-labeled conjugate into 120mM Tris buffer solution with the pH value of 8.2, wherein the volume ratio of the conjugate to the Tris buffer solution is 1: 100-1: 10000.
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| CN109111494A (en) * | 2018-08-30 | 2019-01-01 | 苏州博源医疗科技有限公司 | Derivatives of estradiol, immunogene, antibody, enzyme mark conjugate, detection reagent and preparation method thereof |
| CN109180768B (en) * | 2018-09-06 | 2020-04-17 | 苏州博源医疗科技有限公司 | Estrone derivative, immunogen, antibody, enzyme-labeled conjugate, detection reagent and preparation method thereof |
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