Dissociation reagent for aldosterone detection and aldosterone chemiluminescence detection kit

Abstract

The application discloses a dissociation reagent for aldosterone detection and an aldosterone chemiluminescence detection kit comprising the dissociation reagent. The dissociation reagent for aldosterone detection comprises a buffer, a salt, a stabilizer, a dissociation agent and a surfactant, wherein the dissociation agent is used to dissociate and release the bound aldosterone combined with albumin into free aldosterone. The dissociation reagent for aldosterone detection of the application dissociates and releases the bound aldosterone combined with albumin into free aldosterone through the dissociation agent, so that the detection result contains the bound aldosterone, and the clinical requirement is further met. When the dissociation reagent for aldosterone detection of the application is applied to the chemiluminescence detection kit, the bound aldosterone combined with albumin is dissociated and released into free aldosterone through the dissociation agent, so that the detection result contains the bound aldosterone, and the adrenal secretion capacity of aldosterone can be more completely evaluated from the clinical perspective.

Description

Technical Field

[0001] This invention relates to the field of reagents for medical diagnostic instruments, and in particular to a dissociation reagent for aldosterone detection and an aldosterone chemiluminescence detection kit. Background Technology

[0002] Aldosterone is a steroid hormone secreted by the zona glomerulosa of the adrenal cortex. Its primary function is to act on the kidneys, retaining sodium and excreting potassium. Serum aldosterone concentration is an important indicator for screening and diagnosing diseases such as primary aldosteronism, congenital adrenal hyperplasia, and Liddle syndrome. Elevated aldosterone levels are also a risk factor for cardiovascular events, chronic kidney disease, and metabolic syndrome. Therefore, accurate measurement of aldosterone concentration has significant clinical importance.

[0003] Currently, the main methods for detecting aldosterone concentration include radioimmunoassay, mass spectrometry, and chemiluminescence immunoassay. Among them, chemiluminescence immunoassay has become the mainstream method due to its advantages such as high specificity, high sensitivity, high degree of automation, and convenient operation. In chemiluminescence immunoassay, a luminescent substance or enzyme is first labeled onto an aldosterone antibody. After the aldosterone antibody specifically binds to aldosterone in the blood sample, a chemiluminescent substrate is added. After the reaction, the chemiluminescent substrate releases photons, which are detected by the instrument. Based on the relationship between the light intensity detected by the instrument and a known standard aldosterone concentration sample, the actual concentration of aldosterone in the sample can be accurately detected.

[0004] Under normal circumstances, aldosterone in the blood exists primarily in its bound form, which is bound to albumin, accounting for 60%, while only 40% is in a free state. However, existing chemiluminescence immunoassay kits can only detect free aldosterone because their antibodies can only bind to free aldosterone in the sample. Clinically, to comprehensively evaluate the adrenal glands' ability to secrete aldosterone, it is necessary to detect both free and bound aldosterone concentrations. Therefore, the bound aldosterone must first be dissociated into free aldosterone during the detection process.

[0005] Existing chemiluminescence assay kits lack reagents that dissociate bound aldosterone, thus failing to fully meet clinical needs. Summary of the Invention

[0006] Therefore, it is necessary to provide a dissociation reagent for the detection of aldosterone that can solve the above problems.

[0007] Furthermore, it is necessary to provide an aldosterone chemiluminescence detection kit that includes the dissociation reagent for aldosterone detection described above.

[0008] A dissociation reagent for aldosterone detection includes a buffer, a salt, a stabilizer, a dissociation agent, and a surfactant, wherein the dissociation agent is used to dissociate bound aldosterone to albumin and release it into free aldosterone.

[0009] In one embodiment, the dissociating agent is spironolactone.

[0010] In one embodiment, the mass concentration of the dissociation agent is 0.005 wt% to 0.025 wt%.

[0011] In one embodiment, the stabilizer is EDTA·2Na, and the mass concentration of the stabilizer is 0.05wt% to 0.25wt%.

[0012] In one embodiment, the surfactant is sodium dodecyl sulfate, and the mass concentration of the surfactant is 0.025 wt% to 0.15 wt%.

[0013] In one embodiment, the buffer is a phosphate buffer salt, and the molar concentration of the buffer is 0.025M to 0.15M.

[0014] In one embodiment, the salt is sodium chloride, and the salt has a mass concentration of 0.9 wt%.

[0015] In one embodiment, the pH of the dissociation reagent for aldosterone detection is 6.8 to 7.2, and the pH of the dissociation reagent for aldosterone detection is adjusted by hydrochloric acid or sodium hydroxide.

[0016] In one embodiment, the dissociation reagent for aldosterone detection further includes a preservative;

[0017] The preservative is Proclin 300, and the mass concentration of the preservative is 0.025wt% to 0.15wt%.

[0018] A chemiluminescent aldosterone detection kit, comprising the above-mentioned dissociation reagent for aldosterone detection.

[0019] The aldosterone detection dissociation reagent of the present invention dissociates bound aldosterone bound to albumin into free aldosterone, thereby enabling the detection result to include bound aldosterone and further meeting clinical needs.

[0020] Traditional chemiluminescence immunoassay kits lack a dissociation agent and can only detect free aldosterone. Their results do not include the concentration of bound aldosterone, making it impossible to comprehensively evaluate the adrenal glands' ability to secrete aldosterone from a clinical perspective. The dissociation reagent for aldosterone detection in this invention, when applied to a chemiluminescence immunoassay kit, dissociates bound aldosterone bound to albumin into free aldosterone, thus including bound aldosterone in the detection results. This allows for a more complete evaluation of the adrenal glands' ability to secrete aldosterone from a clinical perspective, overcoming the shortcomings of traditional chemiluminescence immunoassay kits and further meeting clinical needs.

[0021] Preferably, in this invention, the dissociation agent is spironolactone. Spironolactone has a structure similar to aldosterone and is a competitive inhibitor of aldosterone. Spironolactone can competitively bind to and release bound aldosterone from albumin into free aldosterone.

[0022] It should be noted that spironolactone is a diuretic, commonly used to treat edematous diseases and essential aldosteronism, as an adjunct therapy for hypertension, and for the prevention of hypokalemia. Current technology does not utilize these properties of spironolactone to apply it to aldosterone detection, achieving the dissociation and release of bound aldosterone. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0025] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features defined with "first" and "second" may explicitly or implicitly include at least one of the stated features. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0026] The present invention discloses a dissociation reagent for aldosterone detection according to one embodiment, comprising a buffer, a salt, a stabilizer, a dissociation agent, and a surfactant. The dissociation agent is used to dissociate bound aldosterone bound to albumin and release it into free aldosterone.

[0027] The aldosterone detection dissociation reagent of the present invention dissociates bound aldosterone bound to albumin into free aldosterone, thereby enabling the detection result to include bound aldosterone and further meeting clinical needs.

[0028] Traditional chemiluminescence immunoassay kits lack a dissociation agent and can only detect free aldosterone. Their results do not include the concentration of bound aldosterone, making it impossible to comprehensively evaluate the adrenal glands' ability to secrete aldosterone from a clinical perspective. The dissociation reagent for aldosterone detection in this invention, when applied to a chemiluminescence immunoassay kit, dissociates bound aldosterone bound to albumin into free aldosterone, thus including bound aldosterone in the detection results. This allows for a more complete evaluation of the adrenal glands' ability to secrete aldosterone from a clinical perspective, overcoming the shortcomings of traditional chemiluminescence immunoassay kits and further meeting clinical needs.

[0029] Preferably, in this embodiment, the dissociation agent is spironolactone. Spironolactone has a structure similar to aldosterone and is a competitive inhibitor of aldosterone. Spironolactone can competitively bind to and release bound aldosterone from albumin into free aldosterone.

[0030] It should be noted that spironolactone is a diuretic, commonly used to treat edematous diseases and essential aldosteronism, as an adjunct therapy for hypertension, and for the prevention of hypokalemia. Current technology does not utilize these properties of spironolactone to apply it to aldosterone detection, achieving the dissociation and release of bound aldosterone.

[0031] Preferably, in this embodiment, the mass concentration of the dissociation agent is 0.005wt% to 0.025wt%.

[0032] Particularly preferred is that, in this embodiment, the mass concentration of the dissociation agent is 0.01 wt%.

[0033] Preferably, in this embodiment, the stabilizer is ethylenediaminetetraacetic acid disodium dihydrate (EDTA·2Na), and the mass concentration of the stabilizer is 0.05wt% to 0.25wt%.

[0034] Particularly preferred is that, in this embodiment, the mass concentration of the stabilizer is 0.1 wt%.

[0035] Sodium ethylenediaminetetraacetate dihydrate has six coordinating atoms and two water molecules of crystallization. It is an excellent chelating agent with a strong ability to complex various metal ions and separate metals. It can be used as a bleaching and fixing solution in the processing of color photosensitive materials, as well as a dyeing auxiliary agent, a fiber treatment agent, a cosmetic additive, in pharmaceuticals, food, agricultural chemical micro-fertilizer production, a blood stabilizer, a water softener and stabilizer in detergents, a synthetic rubber, a polymerization initiator, and a quantitative analysis agent for heavy metals.

[0036] Disodium ethylenediaminetetraacetate dihydrate, as a stabilizer, can effectively enhance the stability of drugs. The principle is that disodium ethylenediaminetetraacetate can form stable water-soluble chelates with metal ions, which can prevent its own oxidation and improve the stability of drugs during preparation, storage and clinical formulation.

[0037] Preferably, in this embodiment, the surfactant is sodium dodecyl sulfate, and the mass concentration of the surfactant is 0.025 wt% to 0.15 wt%.

[0038] Particularly preferred is that, in this embodiment, the mass concentration of the surfactant is 0.05 wt%.

[0039] Sodium dodecyl sulfate is an anionic surfactant that is readily soluble in water. It has good compatibility with anionic and nonionic surfactants and possesses excellent emulsifying, foaming, penetrating, detergency, and dispersing properties.

[0040] Preferably, in this embodiment, the buffer is a phosphate buffer salt, and the molar concentration of the buffer is 0.025M to 0.15M.

[0041] In a particularly preferred embodiment, the molar concentration of the buffer is 0.05M.

[0042] Preferably, in this embodiment, the salt is sodium chloride, and the salt concentration is 0.9 wt%.

[0043] Preferably, in this embodiment, the pH of the dissociation reagent for aldosterone detection is 6.8 to 7.2, and the pH of the dissociation reagent for aldosterone detection is adjusted by hydrochloric acid or sodium hydroxide.

[0044] In a particularly preferred embodiment, the pH of the dissociation reagent used for aldosterone detection is 7.

[0045] Preferably, in this embodiment, the dissociation reagent for aldosterone detection also includes a preservative.

[0046] The preservative is Proclin 300, with a mass concentration of 0.025wt% to 0.15wt%.

[0047] In a particularly preferred embodiment, the preservative concentration is 0.05 wt%.

[0048] The present invention also discloses an embodiment of an aldosterone chemiluminescent detection kit comprising the above-described dissociation reagent for aldosterone detection.

[0049] The following are specific examples.

[0050] In the specific embodiment, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and Proclin 300 were all purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; EDTA·2Na, spironolactone, and sodium dodecyl sulfate were all purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0051] Example 1

[0052] Preparation of dissociation reagent for aldosterone detection: Weigh 2.399 g of sodium dihydrogen phosphate, 4.259 g of disodium hydrogen phosphate, and 9 g of sodium chloride and dissolve them in 1000 mL of deionized water to obtain a 50 mM PBS buffer. Then add 1 g of EDTA·2Na, 0.1 g of spironolactone, 0.5 g of sodium dodecyl sulfate, and 0.5 mL of Proclin 300. Stir until all components are dissolved, and adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide to obtain the dissociation reagent for aldosterone detection.

[0053] Example 2

[0054] Preparation of dissociation reagent for aldosterone detection: Weigh 2.399 g of sodium dihydrogen phosphate, 4.259 g of disodium hydrogen phosphate, and 9 g of sodium chloride and dissolve them in 1000 mL of deionized water to obtain a 50 mM PBS buffer. Then add 2.5 g of EDTA·2Na, 0.05 g of spironolactone, 1.5 g of sodium dodecyl sulfate, and 0.25 mL of Proclin 300. Stir until all components are dissolved, and adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide to obtain the dissociation reagent for aldosterone detection.

[0055] Example 3

[0056] Preparation of dissociation reagent for aldosterone detection: Weigh 2.399 g of sodium dihydrogen phosphate, 4.259 g of disodium hydrogen phosphate, and 9 g of sodium chloride and dissolve them in 1000 mL of deionized water to obtain a 50 mM PBS buffer. Then add 0.5 g of EDTA·2Na, 0.25 g of spironolactone, 0.25 g of sodium dodecyl sulfate, and 1.5 mL of Proclin 300. Stir until all components are dissolved, and adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide to obtain the dissociation reagent for aldosterone detection.

[0057] Comparative Example 1

[0058] Preparation of control PBS buffer: Weigh 2.399g of sodium dihydrogen phosphate, 4.259g of disodium hydrogen phosphate, and 9g of sodium chloride and dissolve them in 1000mL of deionized water. Adjust the pH to 7.0 with hydrochloric acid or sodium hydroxide to obtain the control PBS buffer.

[0059] Test Example 1

[0060] Thirty plasma samples were collected. 0.8 mL of each sample was taken out with a pipette and divided equally into two centrifuge tubes, 0.4 mL in each tube. 0.1 mL of the aldosterone detection dissociation reagent prepared in Example 1 and the control PBS buffer prepared in Comparative Example 1 were added to each tube and mixed well.

[0061] The samples with the dissociation reagent for aldosterone detection prepared in Example 1 were designated as the experimental group, labeled A1, A2, A3...A15; the samples with the control PBS buffer prepared in Comparative Example 1 were designated as the control group, labeled B1, B2, B3...B15. The samples from both groups were mixed and allowed to stand for 15 minutes before being tested using chemiluminescence immunoassay. The test results are shown in Table 1 below.

[0062] Table 1: Test Results

[0063]

[0064]

[0065] As can be seen from Table 1, the dissociation reagent for aldosterone detection prepared in Example 1 can dissociate bound aldosterone from albumin and release it into free aldosterone.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A dissociation reagent for the detection of aldosterone, characterized in that, It includes buffers, salts, stabilizers, dissociating agents, and surfactants, wherein the dissociating agent is used to dissociate bound aldosterone to albumin and release it into free aldosterone. The dissociation agent is spironolactone, with a mass concentration of 0.005 wt% to 0.025 wt%. The stabilizer is EDTA·2Na, with a mass concentration of 0.05wt% to 0.25wt%. The surfactant is sodium dodecyl sulfate, with a mass concentration of 0.025wt% to 0.15wt%.

2. The dissociation reagent for aldosterone detection according to claim 1, characterized in that, The buffer is a phosphate buffer salt, and the molar concentration of the buffer is 0.025M~0.15M.

3. The dissociation reagent for aldosterone detection according to claim 2, characterized in that, The salt is sodium chloride, and the mass concentration of the salt is 0.9 wt%.

4. The dissociation reagent for aldosterone detection according to claim 3, characterized in that, The pH of the dissociation reagent for aldosterone detection is 6.8 to 7.2, and the pH of the dissociation reagent for aldosterone detection is adjusted by hydrochloric acid or sodium hydroxide.

5. The dissociation reagent for aldosterone detection according to claim 3, characterized in that, The dissociation reagent for aldosterone detection also includes a preservative; The preservative is Proclin 300, and the mass concentration of the preservative is 0.025wt%~0.15wt%.

6. A chemiluminescent detection kit for aldosterone, characterized in that, Includes the dissociation reagent for aldosterone detection as described in any one of claims 1 to 5.

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