Preparation method and application of acridinium ester labeled aldosterone

Abstract

The invention relates to a preparation method and application of acridinium ester labeled aldosterone, aldosterone antigens are modified to obtain groups capable of being combined with acridinium ester, the groups are connected in a chemical bond mode, and the aldosterone antigens are diluted in a chemiluminescent reagent containing protective substances according to a certain concentration in the application process to improve the stability of the aldosterone antigens in the use process. Meanwhile, a large amount of experimental data also shows that the acridinium ester labeled aldosterone chemiluminescent reagent has high correlation with mass spectrometry in detection results when being applied to an aldosterone magnetic particle chemiluminescent kit, and a reliable clinical basis can be provided for diagnosis of aldosterone hypertension and judgment of focus positions clinically; and the method has good popularization value.

Description

[0001] A preparation method and application of acridine ester labeled aldosterone Technical Field

[0003] The present invention relates to the technical field of in vitro diagnostic detection, and in particular to the preparation of acridine ester-labeled aldosterone, which is diluted into a luminescent reagent containing a protective substance to improve its stability during use and applied to an aldosterone chemiluminescent detection kit of an acridine ester system. Background Art

[0005] Aldosterone is a mineralocorticoid hormone and an important component of the renin-angiotensin-aldosterone system. It is an important indicator for auxiliary diagnosis and research of primary and secondary hypertension. Most of the circulating aldosterone is produced by the zona glomerulosa of the adrenal glands, and a small part is synthesized and secreted locally by non-adrenal tissues such as the myocardium, vascular endothelium, brain, and kidneys. In addition to maintaining the balance of water and salt metabolism and blood pressure stability, aldosterone also promotes oxidative stress, causes myocardial fibrosis, and vascular remodeling. It plays an important role in the occurrence and development of many diseases such as cardiac function, collagen synthesis, and myocardial infarction.

[0006] At present, the main aldosterone detection methods on the market are radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) and chemiluminescence assay (CLIA, MCLIA). There are many differences between different detection methods: as for radioimmunoassay, it has the advantages of high sensitivity and little interference from the surrounding environment, but it is also limited by environmental protection, short shelf life, and low degree of automation. Therefore, this method is currently stuck in a bottleneck period of development and faces the situation of being eliminated; from the late 1980s to the 1990s, the enzyme-linked immunosorbent assay (ELISA) gradually emerged. In this method, the detection sensitivity is improved after the enzyme is colored by the substrate, but due to the influence of steric hindrance, the sensitivity and repeatability are still limited to a certain extent; in the late 1990s, the chemiluminescence immunoassay (Chemilµminescence Immµnoassay, CLIA) was valued and promoted. This method has the advantages of being simple and easy to use, easy to prepare markers, high stability, easy to achieve full automation and no pollution to the environment. It is particularly suitable for the requirements of modern clinical testing for automation, accuracy, environmental protection, etc. Therefore, this method is well received by laboratory medicine workers and clinical physicians. The development of mass spectrometry technology in my country started relatively late, and has only gradually emerged since 2000. It has certain advantages in high-end quantitative detection and analysis, and accurate measurement of proteins, nucleotides and sugars, and can be used as an important reference for measurement results. Currently, the magnetic particle chemiluminescence immunoassay method is the mainstream detection method for aldosterone projects.

[0007] As for aldosterone, it is first of all a steroid hormone with a molecular weight of only 360.4. To establish a magnetic microparticle chemiluminescent immunoassay method, the first problem faced is to achieve its connection with acridinium ester (NSP-SA-ADH). If the two are connected at the wrong site, the substance detected will not be aldosterone. As a result, although there are many chemiluminescent kits for detecting aldosterone on the market, the direct test results of each company vary greatly and there are many fakes. The second problem faced is the technical problem of whether the aldosterone molecule can remain stable in a dilute solution for a long time after being modified and connected to the acridinium ester. In short, the stability of the aldosterone luminescent reagent is a key factor related to the quality of the aldosterone detection kit.

[0008] In order to solve the problem of acridinium ester connection, the present invention transforms aldosterone antigen by O-carboxymethylhydroxylamine method to form immune aldosterone oxime, and then directly labels aldosterone oxime and acridinium ester by mixed anhydride method without interference of carrier substances, thereby achieving effective connection between acridinium ester and aldosterone antigen.

[0009] In order to solve the stability problem of acridinium ester linkers, the present invention adds some protective substances to the diluent to greatly extend the inactivation time of the linker, thereby achieving the purpose of improving stability to meet the needs of clinical testing.

[0010] The problem to be solved by the present invention is to achieve effective connection between acridinium ester and aldosterone antigen, and at the same time improve its stability in diluent and improve its accuracy in the field of medical testing, so as to meet the requirements of clinical testing.

[0011] In order to solve the above technical problems, the technical solution adopted by the present invention is to transform the aldosterone molecule to form an oxime group that can be connected to the acridinium ester, further label it with the acridinium ester, dilute the label to a suitable concentration, and form an aldosterone luminescent reagent. The luminescent reagent can be stably stored, further promoting the improvement of the accuracy of aldosterone magnetic particle detection.

[0012] The technical advantages of the present invention are:

[0013] 1) The experimental design is clear; the antigen modification process shows obvious precipitation-dissolution changes and changes in the substance in the liquid phase, indicating the success or failure of the experimental results, making the experimental process easy to judge and control.

[0014] 2) The components of the luminescent reagents are simple and easy to obtain, and are all common chemical reagents in the laboratory. In particular, the main solution contains common reagents such as phosphate buffer, propylene glycol, and glycerol.

[0015] 3) It does not contain corrosive solvents, is pollution-free, safe and environmentally friendly, and will not cause harm to the environment.

[0016] This luminescent reagent has been well applied in the aldosterone (ALD) detection kit (magnetic particle chemiluminescence method), which improves the accuracy of the reagent and shows 100% consistency with the test results of pathological samples of 44 clinically confirmed cases of primary aldosterone. The blood tests on the left and right adrenal glands of 22 cases of the same case with confirmed primary aldosterone showed obvious differences in the results, indicating that the results on the lesion side are consistent with the actual clinical judgment, and have promotion and application value. Summary of the invention

[0018] The invention relates to a preparation method and application of acridinium ester labeled aldosterone.

[0019] A method for labeling aldosterone with an acridinium ester, wherein the labeled acridinium ester-aldosterone is made into a chemiluminescent reagent with stable performance, characterized in that the chemiluminescent reagent comprises: acridinium ester labeled aldosterone, phosphate solution, glycerol, dipotassium salt of ethylenediaminetetraacetic acid, acetaminophen (AP), fish skin gelatin, propylene glycol, and ProClin 300.

[0020] The acridinium ester-labeled aldosterone involved in the present invention is obtained by first modifying aldosterone antigen by O-carboxymethylhydroxylamine method, and the method is: aldosterone antigen reacts with O-carboxymethylhydroxylamine → decompresses and drains → acidifies → extracts → decompresses and drains, and then aldosterone oxime is labeled by mixed acid anhydride method, and the preparation method is: aldosterone oxime reacts with acridinium ester → blows (drains) the label → re-dissolves in methanol → separates by thin layer chromatography → prepares acridinium ester-aldosterone, and the prepared acridinium ester-labeled aldosterone can be diluted into a diluent that can ensure its stability to prepare a luminescent reagent and applied to an aldosterone magnetic particle chemiluminescent detection kit of an acridinium ester luminescent system.

[0021] The luminescent reagent diluent involved in the present invention comprises: phosphate solution, acetaminophen (AP), glycerol, dipotassium salt of ethylenediaminetetraacetic acid, fish skin gelatin, propylene glycol, and ProClin 300; the mass percentage concentrations are respectively 92.15% 0.02 mol / L phosphate solution, 5% glycerol, 0.1% acetaminophen (AP), 0.4% dipotassium salt of ethylenediaminetetraacetic acid, 0.25% fish skin gelatin, 2% propylene glycol, and 0.1% ProClin-300.

[0022] The luminescent reagent of the present invention comprises acridinium ester-labeled aldosterone and a luminescent reagent diluent, and the preparation method thereof is as follows: adding acridinium ester-labeled aldosterone to the luminescent reagent diluent according to a certain volume percentage concentration, mixing, and storing at 2-8° C. for 12 months.

[0023] The preparation method of the luminescent reagent diluent of the present invention is as follows: taking 1 liter as an example, 1.0 g of acetaminophen (AP), 0.04 g of dipotassium ethylenediaminetetraacetate, 25 mg of fish skin gelatin, 50 mL of glycerol, 20 mL of propylene glycol, 100 mL of 0.2 mol / L pH 7.4 PBS-KCl, 764 mL of sodium chloride injection, adjusting the pH to 6.0 with 1 mol / L HCL, and 1 mL of ProClin-300 are weighed on a balance.

[0024] The method for modifying aldosterone antigen described in the present invention is: weigh 3 mg of aldosterone, 1.5 mg of O-carboxymethylhydroxylamine and 3 mg of anhydrous sodium acetate, dissolve them together in 3 mL of methanol, stir them electromagnetically at 37±2° C. for 2 hours, drain them under reduced pressure, add 0.75 mL of 1 mol / L hydrochloric acid (white precipitate appears), place it in a refrigerator for 1 hour, extract it three times with 3 mL of cold ether, drain the extract under reduced pressure, and obtain 2 mg of aldosterone-3 oxime (yellow colloidal powder).

[0025] The method for labeling aldosterone-3-oxime with acridinium ester of the present invention is as follows: weigh 1 mg of aldosterone-3-oxime (aldosterone-3-CMO), add 100 µL of 1,4-dioxane to dissolve, 2 µL of tri-n-butylamine and 2 µL of isobutyl chloroformate, and react at 4°C for 30 minutes. 1 mg of acridinium ester was dissolved in a mixed solution of 50 µL DMF + 50 µL purified water + 10 µL 1 mol / L NaOH. Aldosterone and acridinium ester were mixed in a mass ratio (mg:mg=1:1) and reacted at 2-8°C for 20 h. The marker was blown dry with a nitrogen blower. All the solid matter in the brown bottle was dissolved with 200 µL methanol. The sample was spotted on a silica gel chromatography plate with a capillary tube and blown dry with a hair dryer. Ethyl acetate: ethanol = 5:5 was used as an extender. The silica gel plate with the spotted sample was placed in a chromatography cylinder for sample separation. The relative migration value (Rf) of the target band was about 0.65. The yellow target band silica gel powder was scraped off. An appropriate amount of DMF was added to transfer the marker on the silica gel powder to the liquid phase. The silica gel powder suspension was centrifuged at 5000 rpm for 5 minutes. The supernatant was collected and transferred to a clean brown bottle, which was the acridinium ester-aldosterone marker.

[0026] The concentration of propylene glycol in the luminescent reagent diluent of the present invention can be adjusted to a volume concentration (V / V%) between 5% and 10%. Further increase in concentration affects the accuracy of the liquid injection volume of the automatic luminometer, which should be taken into consideration.

[0027] The present invention further provides the use of the above-mentioned pyridinium ester labeled aldosterone, which has been verified in terms of stability and measurement accuracy of the aldosterone magnetic particle chemiluminescence kit.

[0028] The purity of the chemical reagents used in the diluent of the present invention is analytically pure. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 . Correlation diagram between the luminescent reagent of the present invention and the results of mass spectrometry DETAILED DESCRIPTION

[0032] Example 1: Comparison of the application effects of the present invention in terms of shelf life stability

[0033] Methods: The luminescent reagent was used in the aldosterone (ALD) detection kit (magnetic particle chemiluminescence method). Acridinium ester-labeled aldosterone was prepared into two luminescent reagent diluents, A and B (see Table 1), which were stored at 2-8°C for different periods of time. The luminescent values ​​were regularly tested with the same concentration of standard, antibody solution and magnetic particle reagent to investigate the protective effect of different dilutions on acridinium ester-labeled aldosterone within 1 year. The results are shown in Table 1.

[0034] Table 1 Luminescent reagent dilution instructions

[0035]

[0036] Table 2 The results of storage time and luminescence value change of acridinium ester labeled aldosterone in two dilution solutions A and B

[0037]

[0038]

[0039] From the table data, the luminescence value of the calibrant for the same acridinium ester labeled aldosterone in the A luminescent reagent diluent decreased significantly, and the luminescence count was only about 50% of the original after 12 months. However, the luminescence value in the B luminescent reagent diluent remained at about 87% after 12 months and tended to a stable state, and the correlation coefficient of the curve did not change much, indicating that the luminescent reagent of the present invention has a good protective effect.

[0040] Example 2: Comparison of the effects of labeling acridinium ester and aldosterone using different methods

[0041] Methods: Different methods were used to connect acridinium ester and aldosterone: two labeling and application schemes A and B (see Table 3) were used to detect 50 samples quantified by mass spectrometry using reagents from the two schemes. The results are shown in Table 4.

[0042] Table 3 Dilution Description

[0043]

[0044] Table 4 Comparison of the application effects of different labeling methods (pg / mL)

[0045]

[0046] Table 5 Correlation analysis results between the two labeling methods and mass spectrometry results

[0047]

[0048] From the data in Table 5, it can be intuitively found that the correlation between the measured values ​​of acridinium ester aldosterone labeled with scheme B and mass spectrometry is 0.9236, and the results of the two are highly correlated.

[0049] Example 3: Application effect of the present invention in detecting aldosterone (ALD) samples in a detection kit (magnetic particle chemiluminescence method)

[0050] Method: The luminescent reagent of the present invention was applied to the aldosterone (ALD) detection kit (magnetic particle chemiluminescence method) produced by Beijing North Biotechnology Research Institute Co., Ltd. to detect 44 samples of patients with primary aldosterone (including the same person with different blood sampling locations), and the results were compared with those of mass spectrometry. The results are shown in Table 6:

[0051] Table 6 Results of testing 44 samples of primary aldehyde

[0052]

[0053]

[0054] The data in Table 6 show that the chemiluminescent reagent of the present invention is applied to the aldosterone (ALD) detection kit (magnetic particle chemiluminescence method), and the detection results are highly correlated (r value reaches 0.9006), and are 100% consistent with the actual judgment results of the clinical lesion side of the patient, verifying that the chemiluminescent reagent of the present invention is highly accurate.

[0055] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A preparation method and application of acridinium ester-labeled aldosterone, wherein the preparation method of the acridinium ester-labeled aldosterone is to first modify the aldosterone antigen by the O-carboxymethylhydroxylamine method, wherein the aldosterone antigen reacts with O-carboxymethylhydroxylamine → decompresses and drains → acidifies → extracts → decompresses and drains, and then labels aldosterone oxime by the mixed acid anhydride method, wherein the preparation method is to react aldosterone oxime with acridinium ester → blows (drains) the label → re-dissolves in methanol → separates by thin layer chromatography → prepares acridinium ester-aldosterone, wherein the application characteristic is to dilute the aldosterone into a diluent that can ensure its stability to prepare a luminescent reagent and apply the aldosterone magnetic particle chemiluminescent detection kit of the acridinium ester luminescent system.

2. The use according to claim 1, characterized in that The aldosterone luminescent reagent comprises acridinium ester labeled aldosterone, phosphate solution, glycerol, ethylenediaminetetraacetic acid dipotassium salt, acetaminophen (AP), fish skin gelatin, propylene glycol, and ProClin-300.

3. The luminescent reagent according to claim 1, characterized in that The acridinium ester-labeled aldosterone dilution solution contains 92.15% 0.02 mol / L phosphate solution, 5% glycerol, 0.1% acetaminophen (AP), 0.4% dipotassium ethylenediaminetetraacetic acid, 0.25% fish skin gelatin, 2% propylene glycol, and 0.1% ProClin-300 in mass percentage concentrations.

4. The acridinium ester-labeled aldosterone according to any one of claims 1 to 3, characterized in that The method for labeling aldosterone with acridinium ester is as follows: 3 mg of aldosterone, 1.5 mg of O-carboxymethylhydroxylamine and 3 mg of anhydrous sodium acetate are dissolved in 3 mL of methanol, stirred at 37±2°C for 2 hours, dried under reduced pressure, 0.75 mL of 1 mol / L hydrochloric acid is added (white precipitate appears), placed in a refrigerator for 1 hour, extracted 3 times with 3 mL of cold ether, and the extract is dried under reduced pressure to obtain 2 mg of aldosterone-3 oxime (yellow colloidal powder), 1 mg of aldosterone-3 oxime (aldosterone-3-CMO) is weighed, 100 µL of 1,4-dioxane is added to dissolve, 2 µL of tri-n-butylamine and 2 µL of isobutyl chloroformate are added, and the reaction is carried out at 4°C for 30 minutes, and 1 mg of acridinium ester is treated with 50 µL of DMF+50 µL of purified water+10 µL of 1 mol / L NaOH. The mixed solution was dissolved, aldosterone and acridinium ester were mixed according to the mass ratio (mg:mg=1:1), reacted at 2~8℃ for 20h, the marker was blown dry with a nitrogen blower, and all the solid substances in the brown bottle were dissolved with 200µL methanol. The sample was spotted on the silica gel chromatography plate with a capillary tube, and blown dry with a hair dryer. Ethyl acetate: ethanol = 5:5 was used as an extender, and the silica gel plate with the sample spotted was placed in a chromatography cylinder for sample separation. The relative migration value (Rf) of the target band was about 0.

65. The yellow target band silica gel powder was scraped off, and an appropriate amount of DMF was added to transfer the marker on the silica gel powder to the liquid phase. The above silica gel powder suspension was centrifuged at 5000rpm for 5 minutes, and the supernatant was collected and transferred to a clean brown bottle, which was the acridinium ester-aldosterone marker.

5. A luminescent reagent as claimed in any one of claims 1 to 3, characterized in that The preparation method of the luminescent reagent diluent is as follows: taking 1 liter as an example, use a balance to weigh 1.0 g of acetaminophen (AP), 0.04 g of dipotassium ethylenediaminetetraacetate, 25 mg of fish skin gelatin, 50 mL of glycerol, 20 mL of propylene glycol, 100 mL of 0.2 mol / L pH7.4 PBS-KCL, 764 mL of sodium chloride injection, adjust the pH to 6.0 with 1 mol / L HCL, and 1 mL of ProClin-300.

6. A luminescent reagent according to any one of claims 1 to 3, characterized in that The concentration of glycerol can be adjusted to a volume concentration (V / V%) between 5% and 10%.

7. The use of the pyridinium ester-labeled aldosterone according to any one of claims 1 to 5 has been verified in terms of stability and measurement accuracy of an aldosterone magnetic particle chemiluminescence kit.

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