A urea determination kit with good stability and anti-hemolysis sample interference and a preparation method thereof

Abstract

The application discloses a urea determination kit with good stability and anti-hemolysis sample interference and a preparation method thereof, relates to the field of in-vitro diagnostic reagents, and the urea determination kit comprises reagent R1 and reagent R2, the volume ratio of the two is R1:R2=4:1 when used, the reagent R1 mainly comprises a buffer, a preservative, a stabilizer, an anti-interference agent, alpha-ketoglutaric acid and NADH; and the reagent R2 mainly comprises a buffer, a preservative, a stabilizer, an enzyme protective agent, a tool enzyme, ADP and alpha-ketoglutaric acid. The opening stability of the kit is improved by adding the enzyme protective agents DTT and N-acetylcysteine, and the interference of hemolysis samples on determination is reduced by adding PEI, so that the problems of poor opening stability of urea kits on the market, interference of hemolysis samples on determination, inaccurate detection results and misdiagnosis caused by the problems are solved.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, and more particularly to a urea assay kit with good stability and resistance to interference from hemolyzed samples, and its preparation method. Background Technology

[0002] Urea is the main end product of protein metabolism in the human body, constituting the majority of non-protein nitrogen in the blood. Urea is produced in the liver and excreted into the urine through the kidneys; therefore, urea levels depend on the amount of protein ingested, protein catabolism, and kidney function. Elevated urea concentrations can be caused by kidney failure, excessive protein intake, and other factors.

[0003] The principle of urea determination is as follows: Urea is hydrolyzed into ammonia and carbon dioxide by urease. In the presence of reduced nicotinamide adenine dinucleotide (NADH), ammonia and α-ketoglutarate react under the catalysis of glutamate dehydrogenase to form glutamate, while NADH is simultaneously oxidized to oxidized nicotinamide adenine dinucleotide (NAD+). The rate of NADH reduction is directly proportional to the urea concentration in the sample. Hemolyzed samples interfere with the determination, mainly due to the release of organophosphates and some enzymes from red blood cells caused by severe hemolysis.

[0004] In actual clinical use, urea assay kits all have a common problem: poor stability after opening. When the urea reagent is opened in the reagent compartment of the biochemical analyzer, the oxidative degradation of the tool enzymes glutamate dehydrogenase and urease causes the calibrated K value to continuously increase. This means that clinicians need to recalibrate every once in a while. In severe cases, the reagent may become ineffective. If clinicians do not pay attention, it can easily lead to inaccurate test results and misdiagnosis. Summary of the Invention

[0005] The purpose of this invention is to develop a urea assay kit with good open-pack stability and resistance to interference from hemolyzed samples, as well as its preparation method, to solve the problems of poor open-pack stability and interference from hemolyzed samples in the assay.

[0006] This kit effectively detects urea levels in serum and features good stability after opening and resistance to interference from hemolyzed samples. This invention mitigates interference from hemolyzed samples by adding polyethyleneimine (PEI). PEI significantly degrades adenosine kinase in the erythrocytes of hemolyzed samples. Adenosine kinase is an enzyme that interconverts between ADP and ATP, and this interconversion can interfere with colorimetric reactions. Therefore, adding a certain amount of PEI to the urea reagent can prevent the interconversion between ADP and ATP, thus reducing interference from hemolyzed samples. DTT and N-acetylcysteine ​​protect glutamate dehydrogenase and urease, preventing their oxidative degradation and ensuring good stability of the reagent during use after opening.

[0007] A urea assay kit with good stability and resistance to interference from hemolyzed samples includes reagent R1 and reagent R2. Reagent R1 includes: buffer, preservative, stabilizer, anti-interference agent, α-ketoglutarate, and NADH; reagent R2 includes: buffer, preservative, stabilizer, enzyme protectant, tool enzyme, ADP, and α-ketoglutarate.

[0008] Based on the above scheme, the anti-interference agent of reagent R1 is PEI.

[0009] Based on the above scheme, the enzyme protectant of reagent R2 is one or both of DTT and N-acetylcysteine.

[0010] Based on the above scheme, the tool enzymes of reagent R2 are glutamate dehydrogenase and urease.

[0011] Based on the above scheme, the buffer solutions for reagents R1 and R2 are both Tris buffer; the preservatives for reagents R1 and R2 are both NaN3; the stabilizer for reagent R1 is EDTA Na2, and the stabilizers for reagent R2 are both EDTA Na2 and mannitol.

[0012] Based on the above scheme, preferably, the compositions of reagent R1 and reagent R2 are as follows: Composition of reagent R1:

[0013]

[0014] Composition of reagent R2:

[0015]

[0016] Based on the above scheme, the optimal composition of reagents R1 and R2 is as follows: Composition of reagent R1:

[0017]

[0018]

[0019] Composition of reagent R2:

[0020]

[0021] Based on the above scheme, the volume ratio of reagent R1 and reagent R2 when used is R1:R2 = 4:1.

[0022] A method for preparing a urea assay kit with good stability and resistance to interference from hemolyzed samples, based on 1 L each of reagent R1 and reagent R2.

[0023] The preparation steps for reagent R1 are as follows:

[0024] S1: Weigh an appropriate amount of pure water in a beaker and place it on a magnetic stirrer;

[0025] S2: Add 8g Tris and stir until completely dissolved;

[0026] S3: Add 0.375g EDTA Na2 and stir until completely dissolved;

[0027] S4: Add 0.5g NaN3 and stir until completely dissolved;

[0028] S5: Add 0.75g of α-ketoglutaric acid and stir until completely dissolved;

[0029] S6: Add 2.5g of PEI and stir until completely dissolved;

[0030] S7: Adjust pH to 9.7 (20℃);

[0031] S8: Add 0.32g NADH and stir until completely dissolved;

[0032] S9: Adjust the volume to 1L with pure water;

[0033] It should be noted that during the preparation process, the next ingredient should be added only after the previous ingredient has completely dissolved.

[0034] The preparation steps for reagent R2 are as follows:

[0035] S1: Weigh an appropriate amount of pure water in a beaker and place it on a magnetic stirrer;

[0036] S2: Add 70g Tris and stir until completely dissolved;

[0037] S3: Add 0.48g EDTA Na2 and stir until completely dissolved;

[0038] S4: Add 1g NaN3 and stir until completely dissolved;

[0039] S5: Add 4.93g ADP and stir until completely dissolved;

[0040] S6: Add 3.12g of α-ketoglutaric acid and stir until completely dissolved;

[0041] S7: Add 0.22g mannitol and stir until completely dissolved;

[0042] S8: Add 0.5g DTT and 0.5g N-acetylcysteine, and stir until completely dissolved;

[0043] S9: Adjust pH to 7.9 (20℃);

[0044] S10: Add 5.52 KU of glutamate dehydrogenase and stir until completely dissolved;

[0045] S11: Add 18KU of urease and stir until completely dissolved;

[0046] S12: Make up to 1L with pure water.

[0047] It should be noted that during the preparation process, the next ingredient should be added only after the previous ingredient has completely dissolved.

[0048] The present invention has the following beneficial effects:

[0049] 1. This invention improves the open stability of the reagent kit by adding enzyme protectants DTT and N-acetylcysteine. DTT and N-acetylcysteine ​​can protect glutamate dehydrogenase and urease, prevent them from oxidative degradation, and ensure that the reagent has good stability during the opening and use process.

[0050] 2. This invention reduces the interference of hemolyzed samples on the assay by adding PEI. PEI can greatly degrade adenosine kinase in the red blood cells of hemolyzed samples. Adenosine kinase is an enzyme that interconverts between ADP and ATP, and this interconversion will interfere with the colorimetric reaction. Therefore, adding a certain amount of PEI to the urea reagent can prevent the interconversion between ADP and ATP and reduce the interference of hemolyzed samples.

[0051] 3. The urea assay kit of the present invention has a convenient preparation method, without complicated reaction conditions, and has high production efficiency. Detailed Implementation

[0052] The invention will be further illustrated below with examples:

[0053] The principle of this invention:

[0054] Polyethyleneimine (PEI) is added to mitigate interference from hemolyzed samples. PEI significantly degrades adenosine kinase in the erythrocytes of hemolyzed samples. Adenosine kinase is an enzyme that interconverts between ADP and ATP, and this interconversion can interfere with colorimetric reactions. Therefore, adding a certain amount of PEI to the urea reagent can prevent the interconversion between ADP and ATP, thus reducing interference from hemolyzed samples. DTT and N-acetylcysteine ​​protect glutamate dehydrogenase and urease, preventing their oxidative degradation and ensuring good stability of the reagent during use after opening.

[0055] Example 1: Stability Test Experiment

[0056] I. Experimental Design

[0057] (1) Reagent R1 includes the following components:

[0058]

[0059] (3) The enzyme protectants include two types: DTT (dithiothreitol) and N-acetylcysteine. Each experimental group uses one or both of these enzyme protectants. In addition, this embodiment also includes an experimental group 7 without enzyme protectants. For details of the experimental group settings and dosages, please refer to Table 1:

[0060] Table 1. Combinations and concentrations of enzyme protectants

[0061]

[0062]

[0063] (4) Preparation method

[0064] Preparation of reagent R1

[0065] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add 8g Tris, 0.375g EDTANa2, 0.5g NaN3, and 0.75g α-ketoglutaric acid in sequence. After the raw materials are completely dissolved, adjust the pH to 9.7 (20℃). Finally, add 0.32g NADH. After the raw materials are completely dissolved, bring the volume to 1L with pure water. (Note that during the preparation process, each raw material must be completely dissolved before adding the next.)

[0066] Preparation of reagent R2

[0067] Weigh an appropriate amount of pure water into a beaker and place it on a magnetic stirrer. Add the following in sequence: 70g Tris, 0.48g EDTA Na2, 1g NaN3, 4.93g ADP, 3.12g α-ketoglutarate, 0.22g mannitol, and enzyme protectant (Experimental Group 1: 0.5g DTT, 0.5g N-acetylcysteine; Experimental Group 2: 0g DTT, 0.5g N-acetylcysteine; Experimental Group 3: 0.5g DTT, 0g N-acetylcysteine; Experimental Group 4: 0.25g DTT, 0.5g N-acetylcysteine; Experimental Group 5: 0.5g DTT, 0.25g N-acetylcysteine; Experimental Group 6: 1g DTT, 1g...). N-acetylcysteine; Experimental group 7 (no enzyme protectant added); After the raw materials are completely dissolved, adjust the pH to 7.9 (20℃); then add 5.52 KU of glutamate dehydrogenase and 18 KU of urease in sequence; after the raw materials are completely dissolved, bring the volume to 1 L with pure water. (Note that during the preparation process, the next raw material should be added only after the previous one has completely dissolved.) II. Experimental Methods and Results Analysis

[0068] (1) Experimental methods

[0069] Each experimental group was calibrated and quality controlled on a Hitachi 7180 biochemical analyzer. Five measurements were taken and the average value was recorded. After that, the samples were kept unsealed in the reagent compartment and were not calibrated during the period.

[0070] Parameters measured by Hitachi 7180 Biochemical Analyzer:

[0071]

[0072] (2) Experimental Results and Analysis

[0073] The K value and quality control test value can be read directly from the biochemical analyzer.

[0074] Table 2. K values ​​for the first calibration of each experimental group.

[0075]

[0076]

[0077] Table 3. Quality control measurement results of each experimental group during the opening period (unit: mmol / L)

[0078]

[0079]

[0080] Table 2 shows the K values ​​of experimental groups 1-7 during the first calibration, and Table 3 shows the quality control test results of experimental groups 1-7 during the opening period. As can be seen from the data in Table 3, on day 30 after opening, the quality control test values ​​of experimental groups 1-5 were all within the acceptable range of 2 SD, while the quality control test values ​​of experimental group 6 and experimental group 7 (without added enzyme protectant) were below 2 SD. Therefore, the combined concentration of the two enzyme protectants in experimental group 6 can be ruled out, and it is demonstrated that adding an appropriate amount of enzyme protectant has a stabilizing effect on the quality control test values ​​during the opening period. Comparing the quality control test data of experimental groups 1-5 after 40 days of opening, it can be found that the quality control test value of experimental group 1 is still within the 2 SD range, indicating the best stability after opening.

[0081] Table 4 shows the changes in K values ​​after 30 days in Kaifeng, with the urea detection channel set up on the Hitachi 7180 biochemical analyzer, and after recalibrating experimental groups 1-7.

[0082] Table 4. K values ​​for the second calibration of each experimental group.

[0083]

[0084] The increase in the absolute value of K indicates that the reactivity of the reagent decreases. Experimental verification shows that this is mainly due to the degradation of glutamate dehydrogenase and urease in reagent R2. As shown in the experimental data in Table 4, the ratio of the two enzyme protectants in experimental group 1 can effectively slow down the degradation of these two tool enzymes.

[0085] Therefore, based on the combined data changes of the calibration K value (Tables 2 and 4) and the experimental results of the quality control test during the opening period (Table 3), the optimal combination of enzyme protectants is experimental group 1 (0.5g DTT, 0.5g N-acetylcysteine).

[0086] Example 2: Anti-interference test experiment

[0087] I. Experimental Design

[0088] The experimental design in this embodiment is based on experimental group 1, which showed the best performance in the stability test, and adds the anti-interference agent polyethyleneimine (PEI) to conduct an experiment to test the interference of hemolyzed samples on urea determination.

[0089] (1) Reagent R1 includes the following components:

[0090]

[0091] (2) Reagent R2 includes the following components:

[0092]

[0093]

[0094] (3) Six concentrations of PEI (polyethyleneimine) were set up, and six experimental groups were set up. See Table 5 for details:

[0095] Table 5: PEI concentration settings in each experimental group

[0096] Experimental group 1 (control) 0.00％ 1:1 (0.5g / L:0.5g / L) Experimental group 8 0.05％ 1:1 (0.5g / L:0.5g / L) Experimental group 9 0.10％ 1:1 (0.5g / L:0.5g / L) Experimental group 10 0.25％ 1:1 (0.5g / L:0.5g / L) Experimental group 11 0.50％ 1:1 (0.5g / L:0.5g / L) Experimental group 12 0.60％ 1:1 (0.5g / L:0.5g / L)

[0097] (4) Preparation method

[0098] Referring to Example 1, PEI was added after adding α-ketoglutarate and before adjusting the pH.

[0099] II. Experimental Methods and Results Analysis

[0100] (1) Experimental methods

[0101] Fresh blood samples were divided into two portions and placed in coagulation tubes. One sample was centrifuged after being left at room temperature for 30 minutes. After separating the serum, it was observed by the naked eye that there was no hemolysis, jaundice, or lipemia, and was determined to be a non-hemolytic sample. The other sample was artificially stirred until hemolysis occurred. After centrifugation, it was observed by the naked eye and determined to be a hemolytic sample.

[0102] A relative deviation of urea content between hemolyzed and non-hemolyzed samples within ±10% is acceptable. The relative deviation is calculated as follows:

[0103] Relative deviation = (mean urea in hemolyzed samples - mean urea in non-hemolyzed samples) / mean urea in non-hemolyzed samples × 100%

[0104] (2) Experimental Results and Analysis

[0105] In experimental groups 1 and 8-12, the urea content of hemolyzed and non-hemolyzed samples was tested, respectively. Each sample was tested 5 times, and the average value was taken. Experimental group 1 of Example 1 was used as a blank control. The results are shown in Table 6:

[0106] Table 6. Measurement results of hemolyzed and non-hemolyzed samples (unit: mmol / L)

[0107] Experimental group 1 7.30 6.35 14.96％ Experimental group 8 7.02 6.34 10.73％ Experimental group 9 6.79 6.32 7.44％ Experimental group 10 6.69 6.34 5.52％ Experimental group 11 6.82 6.33 7.74％ Experimental group 12 7.11 6.32 12.50％

[0108] As shown in Table 6, experimental group 1, serving as the blank control, exhibited the largest relative deviation. Therefore, adding PEI can reduce the relative deviation of urea content between hemolyzed and non-hemolyzed samples. Experimental group 8, with a PEI addition of 0.05%, showed a relative deviation greater than 10%. However, the relative deviations of experimental groups 9, 10, and 11 all met the requirements, indicating that the PEI addition in experimental group 8 was insufficient, resulting in poor anti-interference effects. Furthermore, the results of experimental group 12 show that even with a PEI addition of 0.6%, the relative deviation was also greater than 10%, indicating poor anti-interference effects. Therefore, the suitable range for PEI addition is 0.1%-0.5%; the anti-interference effect is best at an addition of 0.25%. Therefore, the optimal anti-interference experimental group is experimental group 10 (PEI content 0.25%).

[0109] The present invention has been described above by way of embodiments, but the present invention is not limited to the specific embodiments described above. Any modifications or variations made based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A urea assay kit with good stability and resistance to interference from hemolyzed samples, characterized in that: Including reagent R1 and reagent R2, The compositions of reagents R1 and R2 are as follows: Composition of reagent R1: Composition of reagent R2:

2. The urea assay kit with good stability and resistance to interference from hemolyzed samples according to claim 1, characterized in that: The compositions of reagents R1 and R2 are as follows: Composition of reagent R1: Composition of reagent R2:

3. A urea assay kit with good stability and resistance to interference from hemolyzed samples according to claim 1 or 2, characterized in that: The volume ratio of reagent R1 and reagent R2 when used is R1:R2=4:

1.

4. A method for preparing a urea assay kit with good stability and resistance to interference from hemolyzed samples, characterized in that: Assuming 1 L each of reagent R1 and reagent R2, The preparation steps for reagent R1 are as follows: S1: Weigh an appropriate amount of pure water in a beaker and place it on a magnetic stirrer; S2: Add 8 g Tris and stir until completely dissolved; S3: Add 0.375 g EDTA Na2 and stir until completely dissolved; S4: Add 0.5 g NaN3 and stir until completely dissolved; S5: Add 0.75 g α-ketoglutaric acid and stir until completely dissolved; S6: Add 2.5 g of PEI and stir until completely dissolved; S7: Adjust the pH to 9.7 at 20℃; S8: Add 0.32 g NADH and stir until completely dissolved; S9: Adjust the volume to 1L with pure water; The preparation steps for reagent R2 are as follows: S1: Weigh an appropriate amount of pure water in a beaker and place it on a magnetic stirrer; S2: Add 70 g Tris and stir until completely dissolved; S3: Add 0.48 g EDTA Na2 and stir until completely dissolved; S4: Add 1 g NaN3 and stir until completely dissolved; S5: Add 4.93 g ADP and stir until completely dissolved; S6: Add 3.12 g of α-ketoglutaric acid and stir until completely dissolved; S7: Add 0.22 g mannitol and stir until completely dissolved; S8: Add 0.5 g DTT and 0.5 g N-acetylcysteine, and stir until completely dissolved; S9: Adjust the pH to 7.9 at 20°C; S10: Add 5.52 KU of glutamate dehydrogenase and stir until completely dissolved; S11: Add 18 KU of urease and stir until completely dissolved; S12: Make up to 1L with pure water.

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