计算溶液所需的质量、体积或浓度。
尿素 (Urea) 含量检测试剂盒 (脲酶波氏, 微量法)
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文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
尿素(Urea)又称碳酰胺(carbamide)，是哺乳动物和某些鱼类体内蛋白质代谢分解的主要含氮终产物，也是目前含氮量最高的氮肥；尿素检测方法大致分为化学方法和酶学方法，后者被认为是间接方法，先经尿素酶(脲酶)分解尿素为铵离子，然后根据波氏反应检测铵离子的生成量。尿素(Urea)检测试剂盒(脲酶波氏微板法)检测原理是尿素酶水解尿素，产生氨和二氧化碳，氨在碱性条件下与苯酚等反应生成蓝色吲哚酚，吲哚酚的生成量与尿素含量呈正比，通过酶标仪测定570 nm处吸光度，该试剂盒可 用于测定人体、动物的血浆、血清、尿液等样品中尿素(旧称尿素氮，BUN)含量，但尿液最好经过处理后再行检测。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
参考标准曲线范围：根据说明书操作步骤采用酶标仪570 nm 测定尿素标准在0、0.2、0.4、0.6、0.8、1、3、5、10、15、20、25 mmol/L 时的吸光度，据此作出其标准曲线如下：
注意：由于试剂批次、仪器设备、操作方法及工作环境等不同，参考范围会有差异，该值仅供参考，对于要求精确计算尿素含量的，可以采用标准曲线进行多点测定；根据测定经验显示，标准品浓度小于0.2 mmol/L 或大于30 mmol/L，标准曲线会有偏差。
组分表
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 100 T | 储存 | 单次测试使用量 |
| U1522045NT | 尿素 (Urea) 含量检测试剂盒 (脲酶波氏, 微量法) (常温组分) | U1522045A | ddH₂O | 无色液体 | 20 mL | 室温 | 请按说明配制 |
| U1522045WT | 尿素 (Urea) 含量检测试剂盒 (脲酶波氏, 微量法) (2-8℃组分) | U1522045B | 脲酶稀释液 | 无色液体 | 3 mL | 2-8℃. | 每次反应需使用14 μL |
| U1522045C | Urea 显色液 | 无色液体 | 10 mL | 2-8℃. 避光. | 每次反应需使用100 μL | ||
| U1522045D | Urea Assay Buffer | 无色液体 | 10 mL | 2-8℃. 避光. | 每次反应需使用100 μL | ||
| U1522045E | 尿素标准(100 mmol/L) | 无色液体 | 1 mL | 2-8℃. 避光. | 请按说明配制 | ||
| U1522045DT | 尿素 (Urea) 含量检测试剂盒 (脲酶波氏, 微量法)(-20℃组分) | U1522045F | 脲酶溶液 | 无色液体 | 1 mL | -20℃. 避光. | 每次反应需使用7 μL |
注：发货时各组分依据储存温度（RT/2‑8℃/-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 6 个月。
自备材料
1. 水浴锅或恒温箱、离心管或小试管
2. 酶标仪、96 孔板
3. 无氨蒸馏水、沸石
操作步骤(仅供参考)
1. 准备样品：
1) 血浆、血清：血浆、血清按照常规方法制备，直接用于该试剂盒的测定，-20℃冻存。
2) 尿液：尿液样品最好处理后测定，方法如下：取0.6 mL尿液样品，加入沸石0.3 g，加入无氨蒸馏水至15 mL，反复震荡数次，吸附尿液中的游离铵盐， 静置后，吸取稀释尿液，所测结果乘以25，如果尿液比较少，可以等比例减少 各试剂的使用量。如果取1 mL尿液样品，应加入沸石0.5 g，加入无氨蒸馏水至25 mL，反复震荡数次，吸附尿液中的游离铵盐，静置后，吸取稀释尿液，所测结果乘以25。
2. 配制标准品工作液：取适量的尿素标准(100 mmol/L)，稀释成梯度尿素标准 (0、0.2、0.4、0.6、0.8、1、3、5、10、15、20、25 mmol/L)，即为标准品工作液，也可取适量的尿素标准(100 mmol/L)，按尿素标准 (100 mmol/L)：ddH2O=1:19 的比例混合，使尿素浓度达到 5 mmol/L，即为标准品工作液-尿素标准(5 mmol/L) 4℃保存，1周有效。
3. 配制脲酶工作液：取适量的脲酶溶液，按脲酶溶液：脲酶稀释液=1:2的比例混合，即为脲酶工作液；4℃避光保存，1月有效。
4. Urea 加样：按照下表设置空白孔、标准孔、测定孔，溶液应按照顺序依次加入，并注意避免产生气泡。如果样品中的 Urea浓度过高，可以减少样品用量或适当稀释后再进行测定。
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5. Urea 测定：充分混匀，37℃水浴20 min，酶标仪测定570 nm处吸光度，空白孔调零，读取各孔吸光度，分别为A 标准、A 测定。
6. 计算：
有2种检测对应的计算方法：
1) 尿素(mmol/L) 根据标准曲线以及公式计算
2) 尿素(mmol/L)=(A 测定/A 标准)×5mmol/L
A 测定=测定孔的吸光度
A 标准=标准孔的吸光度
参考区间
成年人血清尿素：2.9~8.2 mmol/L
注意事项
1. 本实验可测定560和630 nm处的吸光度。
2. 如果没有酶标仪，也可用分光光度计测定，但应注意加入试剂量不同， 相应的检测次数会大大减少。
3. 避免使用铵盐抗凝剂，否则会使结果偏高。
4. 高浓度氟化物可抑制尿素酶，引起结果假性偏低。
5. 采用酶标仪未调零情况下，空白管OD 值一般在0.08~0.18 之间，5 mmol/L标准管参考范围一般在0.35~0.55之间。
6. 以肉眼观察，一般情况下尿素浓度≤1 mmol/L可显淡绿色或淡蓝色，浓度≥2 mmol/L即可显蓝色，浓度≥15 mmol/L即可显深蓝色，一般情况下接近上限比接近下限更准确。
7. 为了您的安全和健康，请穿实验服并戴一次性手套操作。
Urea, also known as carbamide, is the major nitrogen‑containing end‑product of protein catabolism in mammals and certain fish species. It is also the nitrogen‑rich fertilizer with the highest nitrogen content currently available. Urea detection methods are generally divided into chemical and enzymatic approaches. The enzymatic method is regarded as an indirect assay: urea is first decomposed into ammonium ions by urease, and the produced ammonium ions are subsequently quantified via the Berthelot reaction.The detection principle of the Urea Assay Kit (Urease‑Berthelot Microplate Method) is as follows: urease hydrolyzes urea to produce ammonia and carbon dioxide. Under alkaline conditions, ammonia reacts with phenol‑related reagents to form blue indophenol. The yield of indophenol is positively proportional to urea concentration. The absorbance is measured at 560 nm using a microplate reader. This kit is applicable for determining urea (formerly referred to as blood urea nitrogen, BUN) levels in human and animal plasma, serum, urine and other samples. Urine samples are recommended to be pre‑processed prior to measurement. This kit is for research‑only use and shall not be applied for clinical diagnosis or other purposes.
Reference standard curve range: Follow the procedure described in the manual, measure the absorbance of urea standards at concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1, 3, 5, 10, 15, 20, 25 mmol/L with a microplate reader at 570 nm, and generate the corresponding standard curve accordingly：
Note: Reference values may vary owing to reagent batch, instrument equipment, operating procedures and working‑environment conditions, and are provided for reference only. Multi‑point measurement using a standard curve is recommended for accurate urea quantification. Based on assay experience, deviations in the standard curve will occur when standard concentrations are below 0.2 mmol/L or above 30 mmol/L.
Components
| Storage Unit No. | Storage Unit Name | Component No. | Component Name | Appearance | 100 reaction | Storage | Quantity Per Test |
| U1522045NT | Urea Content Assay Kit (Urease Pheby, Micro Method) (RT Components) | U1522045A | ddH₂O | Colorless liquid | 20 mL | Room temperature | As needed |
| U1522045WT | Urea Content Assay Kit (Urease Pheby, Micro Method) (2-8℃ Components) | U1522045B | Urease dilution solution | Colorless liquid | 3 mL | 2-8℃. | 14 μL for 1 reaction |
| | | U1522045C | Urea Coloration Solution | Colorless liquid | 10 mL | 2-8℃. Store in the dark. | 100 μL for 1 reaction |
| | | U1522045D | Urea Assay Buffer | Colorless liquid | 10 mL | 2-8℃. Store in the dark. | 100 μL for 1 reaction |
| | | U1522045E | Urea standard (100 mmol/L) | Colorless liquid | 1 mL | 2-8℃. Store in the dark. | As needed |
| U1522045DT | Urea Content Assay Kit (Urease Pheby, Micro Method) (-20℃ Components) | U1522045F | Urease solution | Colorless liquid | 1 mL | -20℃. Store in the dark. | 7 μL for 1 reaction |
Notes: Each component is individually packaged according to its storage temperature (RT / 2‑8 °C / -20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 6 months from the date of manufacture.
1. Water‑bath or incubator, centrifuge tubes or small test tubes
2. Microplate reader, 96‑well microplate
3. Ammonia‑free distilled water, boiling stones
Procedures (For Reference Only)
1. Sample preparation
1) Plasma and serum: Prepare plasma and serum following routine laboratory protocols. The obtained samples can be directly used for kit measurement and stored frozen at −20 ℃.
2) Urine: Urine samples are preferably pre‑treated before detection. The pretreatment procedure is described below: Take 0.6 mL urine sample, add 0.3 g boiling stones, and top up to 15 mL with ammonia‑free distilled water. Vortex repeatedly to adsorb free ammonium salts in urine. After standing, collect the diluted urine for assay; multiply the measured result by 25 for final calculation. If the urine volume is limited, reduce the volume of each reagent proportionally. For 1 mL urine sample, add 0.5 g boiling stones and make up to 25 mL with ammonia‑free distilled water. Vortex repeatedly to adsorb free ammonium salts. After standing, take the diluted urine for measurement, and multiply the reading by 25.
2. Preparation of standard working solution Dilute appropriate volume of 100 mmol/L urea stock standard solution to prepare serial gradient standards (0, 0.2, 0.4, 0.6, 0.8, 1, 3, 5, 10, 15, 20, 25 mmol/L), which serve as standard working solutions. Alternatively, mix 100 mmol/L urea stock standard with ddH₂O at a volume ratio of 1:19 to obtain 5 mmol/L urea standard working solution. Store at 4 ℃ and use within one week.
3. Preparation of urease working solution Mix urease stock solution with urease diluent at a volume ratio of 1:2 to prepare urease working solution. Store at 4 ℃ protected from light, valid for one month.
4. Urea loading Set blank wells, standard wells and sample‑testing wells according to the table below. Add solutions in the listed order and avoid bubble formation. If the urea concentration in samples is excessively high, reduce sample loading volume or appropriately dilute samples prior to measurement.
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5. Urea measurement: Mix thoroughly, incubate in a water‑bath at 37 ℃ for 20 min. Measure the absorbance at 570 nm using a spectrophotometer, zero the instrument with the blank well, and record the absorbance values denoted as Astandardand Asample.
6. Calculation:
Two calculation methods are available for this assay:
1) Urea (mmol/L): Calculated using the standard curve and corresponding formula.
2) Urea (mmol/L) =(Asample / Astandard) × 5 mmol/L
Where:
Asample: absorbance of the sample well
Astandard: absorbance of the standard well
Reference Range
Serum urea for adults: 2.9‑8.2 mmol/L
Precautions
1. Absorbance can be measured at 560 nm or 630 nm in this assay.
2. A spectrophotometer can be used in place of a microplate reader; however, reagent volumes must be adjusted accordingly and the number of detectable samples will be greatly reduced.
3. Avoid ammonium‑salt‑based anticoagulants, as they will cause falsely elevated results.
4. High‑concentration fluoride inhibits urease activity and leads to falsely low readings.
5. Without zero‑setting on the microplate reader, the OD value of the blank well is typically 0.08‑0.18, and the OD value for the 5 mmol/L standard well generally ranges from 0.35‑0.55.
6. Visual observation: Samples with urea concentration ≤ 1 mmol/L usually show light green or light‑blue color; samples ≥ 2 mmol/L appear blue; samples ≥ 15 mmol/L show dark‑blue color. Assay accuracy is generally higher near the upper limit rather than the lower limit of detection.
7. For personal safety and health, wear a lab coat and disposable gloves during operation.
规格
规格或纯度
BioReagent, 比色法, 适用于分析
英文名称
Urea Content Assay Kit (Urease Pheby, Micro Method)
应用
Colorimetric Assay, 代谢物检测, 细胞代谢, 酶法分析
检测范围
0-25 mmol/L
检测方法
比色法
定量&半定量
定量
检测仪器
酶标仪
检测波长
570mM
重复性
R²＞0.99
回收率
80%-120%
敏感性
Light-sensitive
样本体积
100T
储存与运输
储存条件
2-8°C储存,避光,室温,-20°C储存
运输条件
低温运输,超低温运输
稳定性与储存
各组分在相应的储存条件下保质期6个月。
组分与储存
| 储运单元货号 | 储运单元名称 | 组分货号 | 组分名称 | 物理外观 | 100 reaction | 储存 | 单次测试使用量 |
| U1522045NT | 尿素(Urea)含量检测试剂盒(脲酶波氏, 微量法) (常温组分) | U1522045A | ddH₂O | 无色液体 | 20 mL | 室温 | 请按说明配制 |
| U1522045WT | 尿素(Urea)含量检测试剂盒(脲酶波氏, 微量法) (2-8℃组分) | U1522045B | 脲酶稀释液 | 无色液体 | 3 mL | 2-8℃. | 每次反应需使用14 μL |
| U1522045C | Urea 显色液 | 无色液体 | 10 mL | 2-8℃. 避光. | 每次反应需使用100 μL | ||
| U1522045D | Urea Assay Buffer | 无色液体 | 10 mL | 2-8℃. 避光. | 每次反应需使用100 μL | ||
| U1522045E | 尿素标准(100mmol/L) | 无色液体 | 1 mL | 2-8℃. 避光. | 请按说明配制 | ||
| U1522045DT | 尿素(Urea)含量检测试剂盒(脲酶波氏, 微量法)(-20℃组分) | U1522045F | 脲酶溶液 | 无色液体 | 1 mL | -20℃. Store in the dark. | 每次反应需使用7 μL |
注：发货时各组分依据储存温度（RT/2‑8℃/-20℃）独立包装，差异化温控运输以保障产品性能。同一订单含多温区物料可能分批次送达，请以实际收货为准。收货后按组分表标注温度分类存放，有效期自生产之日起 6 个月。
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F1039959 |
分析证书 | U1522045 |
| ZJ26F1039958 |
分析证书 | U1522045 |
| ZJ26F1039957 |
分析证书 | U1522045 |
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