Pretreatment method of sample for urine aldosterone detection, kit and detection system

Abstract

The invention provides a pretreatment method of a sample for urine aldosterone detection, a kit and a detection system. The pretreatment method of the urine aldosterone detection sample comprises the following steps: incubating a urine sample for 0.5-1 hour at the room temperature of 20-25 DEG C by adopting a reagent containing beta-glucuronidase; the addition amount of the beta-glucuronidase in a system composed of a urine sample and the beta-glucuronidase reagent is greater than 5000 units / mL. According to the pretreatment method, the pretreatment time required by clinical determination of aldosterone in 24 hours of urine can be remarkably shortened, and rapid and full-automatic pretreatment and accurate quantification of a sample can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of detection, and in particular relates to a sample pretreatment method, a kit and a detection system for urine aldosterone detection. Background Art

[0002] Aldosterone (ALD) is a mineralocorticoid hormone mainly synthesized and secreted by the zona glomerulosa of the adrenal cortex. Under the regulation of the renin-angiotensin system, secreted aldosterone can play the role of sodium retention, potassium excretion, and water reabsorption, which is of great significance for maintaining normal water-salt balance in the human body. It is also one of the important indicators for clinical differential diagnosis of primary aldosteronism and other primary or secondary hypertensive diseases. Although there are methods for measuring peripheral blood aldosterone, single-point determination based on blood samples may be affected by factors such as the patient's body position at the time of blood collection, the use of antihypertensive drugs, blood potassium concentration, and circadian rhythm. Therefore, the use of 24 urine samples can overcome the defects of single-point sampling and obtain stable and reliable results that can truly reflect the total secretion level of individual aldosterone. In urine samples, aldosterone metabolites are mainly phase II metabolites processed by the kidneys, including glucuronic acid-bound and sulfate-bound. The existing chemiluminescence methods for detecting 24-hour urine total aldosterone all rely on a pre-treatment method of hydrolysis with a specific concentration of hydrochloric acid to convert bound aldosterone into free aldosterone. This pre-treatment method is time-consuming (18 hours or overnight acid hydrolysis), has strict hydrolysis conditions (temperature set to 30-37°C, suitable pH value for dissociation is 1), and has complex operation steps (needs to be neutralized with a certain proportion of alkaline reagents). Therefore, it is difficult to achieve automated pre-treatment, and the actual processing is still highly dependent on manual operation.

[0003] The development of urine aldosterone test kits currently on the market all rely on the manual addition of a certain concentration of hydrochloric acid for acid hydrolysis and then neutralization to dissociate the bound aldosterone in the urine sample. The steps of a commonly used test kit are as follows:

[0004] (1) Urine hydrolysis: 100 μL of 24-hour urine was manually drawn and mixed with 200 μL of 0.3 M hydrochloric acid, and then placed in a 30°C water bath for hydrolysis for 18 hours;

[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: The acid-hydrolyzed samples were detected using chemiluminescent immunosandwich technology. The relative light intensity (RLU) detected by the photomultiplier tube was proportional to the aldosterone concentration in the sample, and aldosterone was accurately quantified. The calculation is as follows: 24-hour urine aldosterone mass (μg) = instrument test result (pg / mL) × 5 × 24-hour urine volume (mL) × 10 -6 .

[0007] In addition, "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. 2019Apr 15; 1113: 84-90." compared the acid hydrolysis efficiency of mixed urine samples under different incubation conditions and different hydrochloric acid concentrations. Although the study proposed a pretreatment method of incubating with 0.2M hydrochloric acid for 4 hours, the evaluation results of multiple tubes of urine with different concentrations were still limited in evidence based only on a single tube of mixed urine.

[0008] Based on this study and previous reports, it can be found that the current method of using hydrochloric acid hydrolysis to detect bound aldosterone has the following defects: (1) The pretreatment method of hydrochloric acid dissociation is time-consuming, and the traditional method is to incubate for 18 hours or overnight, 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 temperature for acid hydrolysis is usually set at 37°C, and a water bath or thermostat is required; (4) The pretreatment process relies on manual operation, and alkaline reagents need to be used for neutralization after hydrochloric acid acid hydrolysis. The steps are cumbersome and difficult to automate. Summary of the invention

[0009] In order to solve the above problems, the purpose of the present invention is to provide a sample pretreatment method, a kit and a detection system for urine aldosterone detection, which can significantly shorten the pretreatment time required for clinical determination of 24-hour urine aldosterone, and can achieve rapid, fully automatic pretreatment and accurate quantification of samples.

[0010] In order to achieve the above-mentioned object, the present invention provides a sample pretreatment method for urine aldosterone detection, which comprises the following steps: treating the urine sample by incubating the urine sample at room temperature (20-25°C) for 0.5-1h with a reagent containing β-glucuronidase; the amount of the β-glucuronidase added to the system consisting of the urine sample and the β-glucuronidase reagent is >5000 units / mL.

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

[0012] According to a specific embodiment of the present invention, preferably, the composition of the reagent containing β-glucuronidase includes >200000 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 h.

[0015] The present invention also provides a method for detecting urine aldosterone, which comprises the following steps:

[0016] (1) treating a urine sample using the above-mentioned pretreatment method to obtain a sample to be tested;

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

[0018] According to a specific embodiment of the present invention, the above detection method includes the following specific steps: taking a 24h human urine sample, adding an enzyme buffer (for example, containing 5μL IMCSzyme RT enzyme stock solution and 155μL PBS buffer, the content of IMCSzyme RT enzyme stock solution is>200000units / mL), gently mixing for 30s, and leaving at room temperature for 30 minutes to obtain a sample to be tested; subsequently, the sample can be directly tested on the machine, and urine aldosterone can be detected by chemiluminescence immunosandwich technology.

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

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

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

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

[0023] The detection method of the present invention is simple to operate, time-saving, and has sufficient enzymatic hydrolysis. The combined chemiluminescent immunosandwich method can significantly shorten the pretreatment time required for clinical determination of 24-hour urine aldosterone, and realize the automated detection and accurate quantification of the entire process of rapid pretreatment and capture determination of urine samples. The detection method is reliable and verified, and is expected to overcome many deficiencies of existing urine pretreatment; the present invention has the following beneficial effects:

[0024] (1) The present invention adopts efficient β-glucuronidase dissociation pretreatment and linked chemiluminescent immunosandwich technology to achieve rapid and accurate quantification of total aldosterone in 24-hour urine samples, and the pretreatment time is greatly shortened, which makes up for the detection defects of the commonly used hydrochloric acid dissociation method, such as long time consumption, complex operation, and manual dependence;

[0025] (2) Fully automatic sample pre-processing can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the linear evaluation result diagram;

[0027] Figure 2 The graphs show the measurement results under different enzyme reaction systems. DETAILED DESCRIPTION

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

[0029] Example 1

[0030] This embodiment provides a method for detecting urine aldosterone, which comprises 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, >200,000 units / mL) and 155 μL of PBS buffer solution (containing 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride; purchased from Sigma) were mixed to prepare the enzymatic hydrolysis reagent;

[0033] (2) preparing magnetic microparticle chemiluminescent aldosterone assay reagents: 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 24h urine sample, add 160 μL enzyme buffer (i.e. enzymatic reagent, containing 5 μL IMCSzyme RT enzyme and 155 μL buffer), gently mix at room temperature (20-25°C) for 30 seconds, and the sample to be tested after being placed at room temperature for 30 minutes can be directly tested on the instrument (instrument model: Maglumi 2000plus New Industry Fully Automatic Chemiluminescence Analyzer); this step can be operated using the automated pre-treatment 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: 65pg / ml, range: 45.5-84.5pg / ml) and high-point calibrator (analyte target value: 370pg / ml, range: 259-481pg / 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: The enzymatically treated urine sample (test sample) is directly put into the instrument (instrument model: Maglumi 2000plus New Industry Fully Automatic Chemiluminescence Analyzer) for automatic extraction and quantitative detection of analytes. The chemiluminescence immunoassay process is as follows:

[0039] A. The kit (Aldosterone Assay Kit (Magnetic Microparticle Chemiluminescence Method), from Shenzhen New Industry Biomedical Engineering Co., Ltd.) containing magnetic microspheres (coated with anti-aldosterone antibodies (recombinantly expressed in HEK293 cells)), high and low calibrators (containing chemically synthesized aldosterone antigens at different concentrations; analyte target value of the low-point calibrator: 65pg / ml, range: 45.5-84.5pg / ml; analyte target value of the high-point calibrator: 370pg / ml, range: 259-481pg / ml), luminescent marker (ABEI-labeled anti-ALD (recombinantly expressed in HEK293 cells)) and quality control (containing chemically synthesized aldosterone antigens at different concentrations) was transferred to the fully automatic immunoassay system. By measuring the high and low-point calibrators of the kit, the pre-defined master curve of the kit was adjusted to a new, instrument-specific working curve;

[0040] B. Each time the kit is recalibrated, quality control should be performed using quality control products according to the quality control procedures. If the quality control results are within the expected range, sample testing can continue;

[0041] C. 50 μL of the sample treated with enzymatic hydrolysis (the above-mentioned sample to be tested) was mixed and incubated with magnetic microspheres coated with anti-aldosterone antibodies at 37° C. for about 10 minutes. The test substance in the sample, i.e., aldosterone, was combined with the antibody coated with the magnetic microspheres to form an immune complex. After incubation, the unbound substances were removed by magnetic separation and washing;

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

[0043] E. Finally, add the supporting substrate solution (sodium hydroxide + hydrogen peroxide) to start the chemiluminescent reaction and generate a light signal. The relative light intensity (RLU) detected by the photomultiplier tube is in a certain proportional relationship with the aldosterone concentration in the sample, so aldosterone can be accurately quantified.

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

[0045] 1. Precision evaluation: Referring to the CLSI EP15-A3 scheme, 24h urine pools of three levels of high (H), medium (M), and low (L) were prepared, and multiple tubes were packed in parallel and stored at -80°C. Five parallel 24h urines were taken from the three levels of high, medium, and low urine every day, rewarmed at room temperature, and 40μL of urine was accurately drawn for enzymatic hydrolysis (5μL_IMCSzyme RT enzyme stock solution + 155μL buffer), single tube parallel operation, incubated at room temperature 25°C for 30 minutes and then measured (the determination method is the same as in Example 1), and the determination was continued for 5 days. The results are shown in Table 1 (the sample was diluted 5 times), and the intra-batch, inter-batch 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 the CLSI EP6-A document, low-level mixed 24h urine (L) and high-level mixed 24h urine (H) were prepared into 11 gradients of mixed urine according to the 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. The test was performed 3 times from low to high, and the test method was the same as in Example 1, and the results were recorded. The results are shown in Tables 2 and Figure 1 .

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

[0051] sample first Second time The third time Actual detection mean 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): Take ten portions of blank water and incubate for 30 minutes according to the method of Example 1, and then take the average value. The LOD of this method is 5 pg / mL. The results are shown in Table 3 below.

[0053] Table 3 Dilution verification recovery

[0054]

[0055] 4. Carryover: According to the method of Example 1, the high-level urine sample was continuously measured 3 times, and then 3 tubes of blank solution or deionized water were measured. Carryover was calculated as (blank 1-blank 3) / (high value repeat 1-blank 3). The results showed that the carryover rate of high-value samples was less than 3% when the instrument's quantitative upper limit (>2000pg / mL) was exceeded, which met the requirements. The results are shown in Table 4.

[0056] Table 4 Carryover results

[0057]

[0058] Optimize enzymatic conditions

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

[0060] Enzymes from different tissues:

[0061] A.β-Glucuronidase from Helix pomatia, Type H-1, partially purified powder, ≥300,000 units / g solid, Product No.: G0751, (β-glucuronidase from Roman snail; Brand: Sigma-Aldrich, enzyme activity ≥300KU / g);

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

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

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

[0065] Among them, the β-glucuronidase from Roman snail (brand: Sigma-Aldrich) and the β-glucuronidase from bovine liver (brand: Rhawn) both use 0.1M acetic acid and sodium acetate buffer solution (pH=4.60) as enzyme buffer to prepare the enzyme working solution. The genetically modified β-glucuronidase (brand: IMCS) uses the matching enzyme buffer to prepare the enzyme working solution.

[0066] Comparison of hydrochloric acid hydrolysis was added to evaluate the dissociation efficiency of hydrochloric acid with enzymes from various sources in four human 24h urine samples (U1-U4) with different concentrations. Parallel tubes were set up and the experimental design was as follows:

[0067] A. Add 20 μL of Sigma-Aldrich β-glucuronidase buffer (the enzyme content in the system is >6667U / mL) to 40 μL of 24-hour urine, incubate at 37°C for 3.5 hours, and then add 180 μL of water to the original tube;

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

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

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

[0071] E. 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, take out 200 μL, and add 200 μL of diluted sodium hydroxide solution to neutralize.

[0072] Simultaneously, a blank enzyme buffer or pure water is used to replace the treatment to obtain the urine aldosterone background without acid hydrolysis or enzymatic hydrolysis, that is, the free aldosterone value;

[0073] The above operations all resulted in a 6-fold dilution of the sample, and the instrument automatically took 50 μL of the treated sample and determined the aldosterone concentration using the chemiluminescent immunosandwich method (the detection method was the same as in Example 1);

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

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

[0076]

[0077] 2. Comparison of dissociation efficiency with different enzyme amounts and incubation times

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

[0079] Low, medium, and high 24-hour urine samples (L, M, H) were processed as follows:

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

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

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

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

[0084] The aldosterone concentration was detected at the corresponding time points using the chemiluminescent immunosandwich method (the detection method is the same as in Example 1). For concentration values ​​that exceeded the linearity, the final concentration value was obtained by diluting 2 / 4 / 8 times;

[0085] The results are as follows Figure 2As shown ( Figure 2 In the figure, taking L-1 as an example, it indicates that the low-value 24h urine sample was treated with an enzymatic hydrolysis system containing 1μL IMCS-RT enzyme + 159μL buffer (experimental group). It can be seen that when the enzyme buffer volume is controlled at 200μL, the bound aldosterone can be fully dissociated by enzymatic hydrolysis of 40μL 24h urine for 0.5h using 5μL IMCS-RT enzyme + 155μL buffer.

Claims

1. A method for pre-treating a sample for urine aldosterone detection, comprising the following steps: The urine sample was treated with a reagent containing β-glucuronidase by incubation at 20-25°C for 0.5-1h; The amount of β-glucuronidase added to the system consisting of the urine sample and the β-glucuronidase reagent is greater than 5000 units / mL.

2. The pre-treatment method according to claim 1, wherein: The β-glucuronidase includes IMCSzyme-RT glucuronidase.

3. The pre-treatment method according to claim 1, wherein: The reagent containing β-glucuronidase comprises>200000 units / mL of IMC Szyme-RT β-glucuronidase stock solution and PBS buffer.

4. The pre-treatment method according to claim 1, wherein: The urine sample includes human urine or artificial simulated urine.

5. The pre-treatment method according to claim 1, wherein: The incubation time is 0.5h.

6. A method for detecting urine aldosterone, comprising the following steps: (1) treating a urine sample using the pretreatment method according to any one of claims 1 to 5 to obtain a sample to be tested; (2) The aldosterone content in the sample was determined by chemiluminescent immunosandwich method.

7. A urine aldosterone detection kit, comprising a reagent bottle A containing a β-glucuronidase stock solution and a PBS buffer solution.

8. The urine aldosterone detection kit according to claim 7, wherein: The reagent bottle A contains >200,000 units / mL of IMCSzyme RT β-glucuronidase stock solution.

9. A urine aldosterone detection system, wherein: The urine aldosterone detection system comprises a urine sample pretreatment reagent and a chemiluminescent immunosandwich detection reagent, wherein the urine sample pretreatment reagent comprises an IMCSzyme RTβ-glucuronidase stock solution of >200,000 units / mL and a PBS buffer solution.

10. The urine aldosterone detection system according to claim 9, wherein: The chemiluminescent immunosandwich detection reagent comprises: magnetic microspheres coated with ALD antibodies, PBS buffer, ALD antigen calibrator, Tris-HCl buffer, ABEI-labeled anti-ALD antibody luminescent marker, and substrate solution, wherein the substrate solution comprises sodium hydroxide and hydrogen peroxide.

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