A sample pretreatment method, kit, and detection system for urinary aldosterone detection

Abstract

This invention provides a sample pretreatment method, kit, and detection system for urinary aldosterone detection. The sample pretreatment method for urinary aldosterone detection includes the following steps: incubating the urine sample with a reagent containing β-glucuronidase at room temperature (20-25°C) for 0.5-1 h; the amount of β-glucuronidase added to the system consisting of the urine sample and the β-glucuronidase reagent is >5000 units / mL. This pretreatment method can significantly shorten the pretreatment time required for clinical determination of 24-hour urinary aldosterone, enabling rapid, fully automated sample pretreatment and accurate quantification.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, specifically relating to a sample pretreatment method, reagent kit, and detection system for urinary aldosterone detection. Background Technology

[0002] Aldosterone (ALD) is a mineralocorticoid hormone primarily synthesized and secreted by the zona glomerulosa of the adrenal cortex. Under the regulation of the renin-angiotensin system, secreted aldosterone plays a role in sodium retention, potassium excretion, and water reabsorption, significantly contributing to maintaining normal water and electrolyte balance in the body. It is also a crucial indicator for the clinical differential diagnosis of primary aldosteronism and other primary or secondary hypertension. Although methods for measuring peripheral blood aldosterone exist, single-point measurements based on blood samples can be affected by factors such as patient position during blood collection, antihypertensive medication use, serum potassium concentration, and circadian rhythm. Therefore, 24-hour urine sample testing overcomes the limitations of single-point sampling, obtaining stable and reliable results that accurately reflect an individual's total aldosterone secretion level. In urine samples, aldosterone metabolites are mainly two-phase metabolites processed by the kidneys, including glucuronide-bound and sulfate-bound forms. Current chemiluminescent methods for detecting total aldosterone in 24-hour urine all rely on pretreatment with hydrochloric acid of a specific concentration to convert bound aldosterone into free aldosterone. This pretreatment method is time-consuming (18 hours or overnight acid hydrolysis), requires stringent hydrolysis conditions (temperature set at 30-37℃, suitable pH for dissociation at 1), and involves complex procedures (needing neutralization with a certain proportion of alkaline reagent). Therefore, automating the pretreatment process is difficult, and actual processing still heavily relies on manual operation.

[0003] Currently, commercially available urine aldosterone test kits all rely on a manual pretreatment method involving the addition of a certain concentration of hydrochloric acid for acid hydrolysis followed by neutralization to dissociate bound aldosterone from the urine sample. An example of the steps for a commonly used test kit is shown below:

[0004] (1) Urine hydrolysis: Manually aspirate 100 μL of 24h urine and mix thoroughly with 200 μL of 0.3M hydrochloric acid, then place in a 30℃ water bath for 18 hours for hydrolysis;

[0005] (2) Urine neutralization: Manually aspirate 200 μL of the hydrolyzed sample, add 200 μL of sodium hydroxide solution, mix thoroughly, and complete the urine treatment process;

[0006] (3) Sample Detection: Acid-hydrolyzed samples were detected using chemiluminescent immunoassay sandwich technology. The relative light intensity (RLU) detected by the photomultiplier tube was proportional to the aldosterone concentration in the sample, allowing for accurate quantification of aldosterone. The calculation was as follows: 24-hour urinary aldosterone mass (μg) = Instrument detection result (pg / mL) × 5 × 24-hour urine volume (mL) × 10 -6 .

[0007] Furthermore, the study "Establishment of a rapid and simple liquid chromatography-tandem mass spectrometry method for measuring aldosterone in urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2019 Apr 15; 1113:84-90" compared the acidolysis efficiency of mixed urine samples under different incubation conditions and hydrochloric acid concentrations. Although this study proposed a pretreatment method of incubation with 0.2M hydrochloric acid for 4 hours, the results were based only on a single tube of mixed urine, and the evidence for evaluations of multiple tubes of urine with different concentrations remains limited.

[0008] Based on this study and previous reports, the following drawbacks can be found in the current method of detecting bound aldosterone using hydrochloric acid hydrolysis: (1) The pretreatment method of hydrochloric acid dissociation is time-consuming, and traditionally, 18 hours or overnight incubation is used, especially for samples with high aldosterone concentrations; (2) The acid hydrolysis conditions are harsh and the acid hydrolysis efficiency is low. The theoretical optimal pH value for acid hydrolysis of bound aldosterone is 1; (3) The suitable acid hydrolysis temperature is usually set at 37°C, which requires the use of a water bath or thermostat; (4) The pretreatment process depends on manual operation, and after hydrochloric acid hydrolysis, it is necessary to continue neutralization with alkaline reagents, which is cumbersome and difficult to automate. Summary of the Invention

[0009] To address the aforementioned problems, the present invention aims to provide a sample pretreatment method, reagent kit, and detection system for urinary aldosterone detection. This pretreatment method can significantly shorten the pretreatment time required for clinical determination of 24-hour urinary aldosterone, and can achieve rapid, fully automated sample pretreatment and accurate quantification.

[0010] To achieve the above objectives, the present invention provides a sample pretreatment method for urinary aldosterone detection, comprising the following steps: incubating the urine sample with a reagent containing β-glucuronidase at room temperature (20-25℃) for 0.5-1h; wherein the amount of β-glucuronidase added to the system consisting of the urine sample and the reagent containing β-glucuronidase is >5000 units / mL.

[0011] According to a specific embodiment of the present invention, preferably, the β-glucuronidase includes IMCSzyme-RT glucuronidase.

[0012] According to a specific embodiment of the present invention, preferably, the reagent containing β-glucuronidase comprises >200,000 units / mL of IMCSzyme-RT β-glucuronidase stock solution and PBS buffer.

[0013] According to a specific embodiment of the present invention, preferably, the urine sample includes human urine or artificial simulated urine.

[0014] According to a specific embodiment of the present invention, preferably, the incubation time is 0.5 hours.

[0015] This invention also provides a method for detecting urinary aldosterone, comprising the following steps:

[0016] (1) The urine sample was processed using the above pretreatment method to obtain the sample to be tested;

[0017] (2) The aldosterone content in the sample was determined by chemiluminescent immunoassay sandwich method.

[0018] According to a specific embodiment of the present invention, the above detection method includes the following specific steps: take a 24-hour urine sample, add an enzyme buffer (e.g., containing 5 μL of IMCSzyme RT enzyme stock solution and 155 μL of PBS buffer, with the IMCSzyme RT enzyme stock solution having a concentration of >200,000 units / mL), gently mix for 30 seconds, and incubate at room temperature for 30 minutes to obtain the sample to be tested; subsequently, it can be directly tested using chemiluminescent immunoassay sandwich technology to detect urinary aldosterone.

[0019] The present invention also provides a urinary aldosterone detection kit, the kit comprising reagent bottle A, wherein reagent bottle A contains β-glucuronidase stock solution and PBS buffer solution.

[0020] According to a specific embodiment of the present invention, preferably, the reagent bottle A contains >200,000 units / mL of IMCSzyme RTβ-glucuronidase stock solution.

[0021] The present invention also provides a urinary aldosterone detection system, wherein the urinary aldosterone detection system includes a urine sample pretreatment reagent and a chemiluminescent immunoassay sandwich detection reagent, wherein the urine sample pretreatment reagent includes >200,000 units / mL of IMCSzyme RT β-glucuronidase stock solution and PBS buffer solution.

[0022] According to a specific embodiment of the present invention, preferably, the chemiluminescent immunoassay sandwich assay reagent includes magnetic microspheres (containing ALD antibody coated (recombinantly expressed in HEK293 cells)), PBS buffer, calibrators (different concentrations of ALD antigen), Tris-HCl buffer, luminescent label (ABEI-labeled anti-ALD antibody (recombinantly expressed in HEK293 cells)), and substrate solution (sodium hydroxide + hydrogen peroxide).

[0023] The detection method of this invention is simple to operate, time-saving, and provides thorough enzymatic digestion. Combined with chemiluminescent immunoassay sandwich method, it can significantly shorten the pretreatment time required for clinical determination of 24-hour urinary aldosterone, achieving automated detection and accurate quantification of the entire process from rapid pretreatment to capture and determination of urine samples. This detection method is reliable and validated, and is expected to overcome many shortcomings of existing urine pretreatment methods. This invention has the following beneficial effects:

[0024] (1) The present invention uses a highly efficient β-glucuronidase dissociation pretreatment and a chemiluminescent immunoassay sandwich technology to achieve rapid and accurate quantification of total aldosterone in 24-hour urine samples. The pretreatment time is increased and shortened, which makes up for the detection defects of the existing commonly used hydrochloric acid dissociation method, such as long time consumption, complicated operation and manual dependence.

[0025] (2) It can realize fully automated sample preprocessing. Attached Figure Description

[0026] Figure 1 This is a graph showing the linear evaluation results;

[0027] Figure 2 The graph shows the measurement results under different enzyme reaction systems. Detailed Implementation

[0028] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0029] Example 1

[0030] This embodiment provides a method for detecting urinary aldosterone, which includes the following steps:

[0031] 1. Reagent preparation

[0032] (1) Preparation of enzymatic hydrolysis reagent: 5 μL of modified β-glucuronidase (IMCSzyme RT enzyme, purchased from IMCSzyme, >200000 units / mL) and 155 μL of PBS buffer solution (containing 0.01M phosphate buffer, 0.0027M potassium chloride and 0.137M sodium chloride; purchased from Sigma) were mixed to prepare the enzymatic hydrolysis reagent;

[0033] (2) Prepare the reagents for the magnetic microparticle chemiluminescence method for aldosterone determination: magnetic microspheres coated with anti-ALD antibody, anti-ALD antibody labeled with luminescent marker N-(4-aminobutyl)-N-ethylisoluminol (ABEI), PBS buffer, and Tris-HCl buffer.

[0034] 2. Sample preparation

[0035] Take 40 μL of human 24-hour urine sample, add 160 μL of enzyme buffer (i.e., enzyme digestion reagent, containing 5 μL LMCSzyme RT enzyme and 155 μL buffer), gently mix at room temperature (20-25℃) for 30 seconds, and let stand at room temperature for 30 minutes before testing. The sample can then be directly tested using the Maglumi 2000plus New Industries Fully Automated Chemiluminescence Analyzer. This step can be performed using an automated sample loading system.

[0036] 3. Instrument calibration and testing

[0037] (1) The instrument needs to be calibrated before each use: use the matching low-point calibrator (analyte target value: 65 pg / ml, range: 45.5-84.5 pg / ml) and high-point calibrator (analyte target value: 370 pg / ml, range: 259-481 pg / ml) for calibration. Subsequent measurements can be carried out if the measured value is within the target value range.

[0038] (2) Quantitative detection of urine samples: Urine samples treated with enzyme hydrolysis (samples to be tested) were directly fed into the instrument (Magali 2000plus New Industries Fully Automated Chemiluminescence Analyzer) for automated analyte extraction and quantitative detection. The chemiluminescence immunoassay process is as follows:

[0039] A. Transfer the kit (Aldosterone Assay Kit (Magnetic Microparticle Chemiluminescence Assay), sourced from Shenzhen New Industries Biomedical Engineering Co., Ltd.) containing magnetic microspheres (coated with anti-aldosterone antibody (recombinantly expressed in HEK293 cells)), high and low calibrators (containing different concentrations of chemically synthesized aldosterone antigen; analyte target value of low-point calibrator: 65 pg / ml, range: 45.5-84.5 pg / ml; analyte target value of high-point calibrator: 370 pg / ml, range: 259-481 pg / ml), luminescent label (ABEI-labeled anti-ALD (recombinantly expressed in HEK293 cells)), and quality control (containing different concentrations of chemically synthesized aldosterone antigen) to the fully automated immunoassay system. By measuring the high and low point calibrators of the kit, adjust the predefined master curve of the kit to a new, instrument-specific working curve.

[0040] B. After each kit recalibration, quality control should be performed using quality control materials according to the quality control procedures. If the quality control results are within the expected range, sample testing can continue.

[0041] C. Mix 50 μL of the enzymatically digested sample (the above-mentioned test sample) with magnetic microspheres coated with anti-aldosterone antibody at 37°C and incubate for about 10 minutes. The analyte in the sample, namely aldosterone, binds with the antibody coated with magnetic microspheres to form an immune complex. After incubation, unbound substances are removed by magnetic separation and washing.

[0042] D. Continue to add the antibody labeled with the luminescent marker N-(4-aminobutyl)-N-ethylisoluminol (ABEI) to the magnetic microspheres to form an immune sandwich complex. After incubation, remove unbound substances by magnetic separation and washing.

[0043] E. Finally, add the matching substrate solution (sodium hydroxide + hydrogen peroxide) to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) detected by the photomultiplier tube is proportional to the aldosterone concentration in the sample, thus accurately quantifying aldosterone.

[0044] Methodological validation of a fully automated method for determining aldosterone in urine samples

[0045] 1. Precision Evaluation: Following the CLSI EP15-A3 protocol, 24-hour urine pools at three levels (high (H), medium (M), and low (L)) were prepared, aliquoted into multiple tubes, and stored at -80°C. Each day, five parallel 24-hour urine samples were collected from each of the three levels, warmed to room temperature, and 40 μL of urine was accurately aspirated for enzymatic digestion (5 μL IMCSzyme RT enzyme stock solution + 155 μL buffer). Single-tube parallel operations were performed, incubated at 25°C for 30 minutes, and then measured (measurement method as in Example 1). Measurements were performed continuously for 5 days. Results are shown in Table 1 (samples were diluted 5-fold). Intra-assay, inter-assay, and total imprecision (CV) were 3.2%–4.8%, 3.4%–6.2%, and 3.5%–6.5%, respectively.

[0046] Table 1 Precision verification results

[0047]

[0048]

[0049] 2. Linearity Evaluation: Referring to CLSI EP6-A, low-level mixed 24-hour urine (L) and high-level mixed 24-hour urine (H) were prepared into 11 gradient mixed urine solutions according to volume ratios of L, 0.9L:0.1H, 0.8L:0.2H, 0.7L:0.3H, 0.6L:0.4H, 0.5L:0.5H, 0.4L:0.6H, 0.3L:0.7H, 0.2L:0.8H, 0.1L:0.9H, and H. Each gradient was tested three times from low to high, using the same method as in Example 1, and the results were recorded. The results are shown in Table 2 and... Figure 1 .

[0050] Table 2. Linearity evaluation results (unit: pg / mL)

[0051] sample first The second The third Actual test average Theoretical value Bias % L 44.04 44.03 39.62 42.6 42.6 -0.1％ 0.9L:0.1H 161.3 162.7 159.4 161.1 212.33 -24.1％ 0.8L:0.2H 350.8 338 337.2 342.0 382.06 -10.5％ 0.7L:0.3H 507.1 496.6 482.1 495.3 551.79 -10.2％ 0.6L:0.4H 658.2 652.5 647.7 652.8 721.52 -9.5％ 0.5L:0.5H 820 846.9 832.4 833.1 891.25 -6.5％ 0.4L:0.6H 1059.9 1020.5 1036.6 1039.0 1060.98 -2.1％ 0.3L:0.7H 1205.5 1243.1 1317.9 1255.5 1230.71 2.0％ 0.2L:0.8H 1528.8 1373.9 1532.3 1478.3 1400.44 5.6％ 0.1L:0.9H 1533.6 1570 1655 1586.2 1570.17 1.0％ H 1582 1802.1 1835.5 1739.9 1739.9 0.0％

[0052] 3. Limit of Detection (LOD): Ten blank water samples were incubated for 30 minutes according to the method in Example 1, and the average value was taken. The LOD of this method was 5 pg / mL. The results are shown in Table 3 below.

[0053] Table 3. Dilution Validation Recovery Rate

[0054]

[0055] 4. Carryover contamination: Following the method in Example 1, high-value urine samples were measured three times consecutively, followed by measurements of three blank solutions or deionized water. Carryover contamination was calculated as (blank 1 - blank 3) / (high-value replicate 1 - blank 3). The results showed that even when the high-value samples exceeded the instrument's upper limit of quantitation (>2000 pg / mL), the carryover contamination rate was less than 3%, meeting the requirements. The results are shown in Table 4.

[0056] Table 4 Results of Carrying Pollution

[0057]

[0058] Optimize enzymatic hydrolysis conditions

[0059] 1. Compare the effects of different enzymes and hydrochloric acid hydrolysis on the treatment.

[0060] Enzymes from different tissue sources:

[0061] A. β-Glucuronidase from Helix pomatia, Type H-1, partially purified powder, ≥300,000 units / g solid, catalog number: G0751 (β-glucuronidase from Helix pomatia; brand: Sigma-Aldrich, enzyme activity ≥300 KU / g);

[0062] B. β-glucuronidase derived from bovine liver (brand: Rhawn, enzyme activity ≥1000KU / g);

[0063] C.IMCSzyme, catalog number: 04-ESF3S-005, β-glucuronidase (brand: IMCS, IMCSzyme-3S, enzyme activity >100kU / mL);

[0064] D.IMCSzyme RT, catalog number: 04-RT-005, β-glucuronidase (brand: IMCS, IMCSzyme RT, enzyme activity > 200kU / mL);

[0065] For both the β-glucuronidase derived from Roman snails (brand: Sigma-Aldrich) and the β-glucuronidase derived from bovine liver (brand: Rhawn), the enzyme working solution was prepared using 0.1M acetic acid and sodium acetate buffer solution (pH=4.60) as the enzyme buffer, following the instructions. For the genetically modified β-glucuronidase (brand: IMCS), the enzyme working solution was prepared using the corresponding enzyme buffer.

[0066] To evaluate the dissociation efficiency of hydrochloric acid and enzymes from various sources in four human 24-hour urine samples (U1-U4) at different concentrations, a comparison of hydrochloric acid hydrolysis methods was added. The experimental design was as follows:

[0067] A. Add 20 μL of Sigma-Aldrich β-glucuronidase buffer (enzyme concentration >6667 U / mL) to 40 μL of 24h urine, incubate at 37℃ for 3.5h, and then add 180 μL of water to the original tube;

[0068] B. Add 20 μL of Rhawn β-glucuronidase buffer (enzyme concentration in the system is >6667 U / mL) to 40 μL of 24h urine, incubate at 37℃ for 3.5h, and then add 180 μL of water to the original tube;

[0069] C. Add 155 μL of IMCS-3S enzyme buffer (containing 5 μL of IMCS-3S enzyme and 150 μL of buffer; the enzyme content in the system is >3094 U / mL) to 31 μL of 24h urine, incubate at 55℃ for 3.5h, and then detect.

[0070] D. Add 155 μL of IMCS-RT enzyme buffer (containing 5 μL of IMCS-RT enzyme and 150 μL of buffer; the enzyme content in the system is >5376 U / mL) to 31 μL of 24h urine, incubate at 55℃ for 3.5h, and then detect.

[0071] Add 200 μL of 0.3 M HCl to 100 μL of 24-hour urine, incubate in a 37°C water bath for 18 hours, remove 200 μL, and then add 200 μL of diluted sodium hydroxide solution to neutralize.

[0072] Simultaneously, blank enzyme buffer or pure water was used as a substitute to obtain the background of urinary aldosterone without acid or enzymatic hydrolysis, i.e., the value of free aldosterone.

[0073] All the above operations resulted in a 6-fold dilution of the sample. The instrument automatically took 50 μL of the processed sample and determined the aldosterone concentration using a chemiluminescent immunoassay sandwich method (the detection method is the same as in Example 1).

[0074] The results are shown in Table 5 below. The two genetically modified β-glucuronidases showed superior glucuronic acid dissociation characteristics.

[0075] Table 5. Raw data on aldosterone determination in 24-hour urine samples before and after acid / enzymatic digestion.

[0076]

[0077] 2. Compare the dissociation efficiency with different enzyme amounts and different incubation times.

[0078] β-glucuronidase (IMCS-RT) was selected to optimize the enzyme dosage and hydrolysis time. The experimental design is as follows:

[0079] Urine samples with low, medium, and high values ​​(L, M, H) were processed as follows:

[0080] A. Add 160 μL of IMCS-RT enzyme buffer (containing 1 μL of IMCS-RT enzyme and 159 μL of buffer) to 40 μL of 24-hour urine.

[0081] B. Add 160 μL of IMCS-RT enzyme buffer (containing 3 μL of IMCS-RT enzyme and 157 μL of buffer) to 40 μL of 24-hour urine.

[0082] C. Add 160 μL of IMCS-RT enzyme buffer (containing 5 μL of IMCS-RT enzyme and 155 μL of buffer) to 40 μL of 24-hour urine.

[0083] Incubate at room temperature at 9 time points: 0h, 0.25h, 0.5h, 1h, 2h, 2.5h, 3h, 3.5h, and 4h.

[0084] The concentration of aldosterone was detected at the corresponding time points using a chemiluminescent immunoassay sandwich method (detection method is the same as in Example 1). For concentration values ​​that exceeded the linear range, the final concentration values ​​were obtained by dilution by 2 / 4 / 8 times.

[0085] The results are as follows Figure 2As shown ( Figure 2 In the example of L-1, it represents the experimental group treated with an enzymatic digestion system containing 1 μL LMCS-RT enzyme + 159 μL buffer for a low-value 24h urine sample. It can be seen that when the enzyme buffer volume is controlled at 200 μL, using 5 μL LMCS-RT enzyme + 155 μL buffer to digest 40 μL of 24h urine for 0.5h can fully dissociate bound aldosterone.

Claims

1. A sample pretreatment method for urinary aldosterone detection, comprising the following steps: Urine samples were treated by incubating at 20-25°C for 0.5-1 h with a reagent containing IMCSzyme-RT glucuronidase. The amount of IMCSzyme-RT glucuronidase added to the system consisting of urine sample and IMCSzyme-RT glucuronidase reagent is >5000 units / mL. The reagent containing IMCSzyme-RT glucuronidase comprises >200,000 units / mL of IMCSzyme-RT glucuronidase stock solution and PBS buffer.

2. The preprocessing method according to claim 1, wherein, The urine sample includes human urine or artificially simulated urine.

3. The preprocessing method according to claim 1, wherein, The incubation time is 0.5 hours.

4. A method for detecting urinary aldosterone, comprising the following steps: (1) The urine sample is processed by the pretreatment method according to any one of claims 1-3 to obtain the sample to be tested; (2) The aldosterone content in the sample was determined by chemiluminescent immunoassay sandwich method.

5. A urinary aldosterone detection kit, the kit comprising reagent bottle A, wherein reagent bottle A contains >200,000 units / mL of IMCSzyme RT glucuronidase stock solution and PBS buffer solution.

6. A urinary aldosterone detection system, wherein, The urinary aldosterone detection system includes urine sample pretreatment reagents and chemiluminescent immunoassay sandwich detection reagents. The urine sample pretreatment reagents include IMCSzyme RT glucuronidase stock solution with a concentration of >200,000 units / mL and PBS buffer solution.

7. The urinary aldosterone detection system according to claim 6, wherein, The chemiluminescent immunoassay sandwich assay reagent includes: magnetic microspheres coated with ALD antibody, PBS buffer, ALD antigen calibrator, Tris-HCl buffer, ABEI-labeled anti-ALD antibody luminescent label, and substrate solution, wherein the substrate solution includes sodium hydroxide and hydrogen peroxide.

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