尿酸 (UA) 是鸟类和爬行类动物的主要代谢产物，正常人体尿液中产物主要为尿素，含少量尿酸。此外，UA还是重要的抗氧化剂，能清除超氧化物，羟自由基等。体内UA生成量和排泄量不平衡会导致多种疾病的发生。例如，血中UA升高会引起痛风、肾功能损害和动脉硬化，相反UA降低会引起恶性贫血，在临床诊断上具有重要的意义。

本试剂盒检测原理：尿酸酶催化尿酸(UA) 生成尿囊素、CO₂及 H₂O₂，H₂O₂ 在过氧化物酶作用下进一步与Amplex Red反应生成试卤灵(Resorufin)，通过检测试卤灵的荧光强度或吸光度来最终计算样品中尿酸的含量。

A1524825	组件	物理外观	100 T	500T	储存条件	单次测试使用量
A1524825A	尿酸酶	液体	0.4 mL	2.0 mL	-20℃.避光.	4 µL
A1524825B	过氧化物酶	液体	0.2 mL	1.0 mL	-20℃.避光.	2 µL
A1524825C	提取液	液体	20 mL	50 mL	-20℃.	按需
A1524825D	反应缓冲液	液体	30 mL	100 mL	-20℃.	100 µL
A1524825E	Amplex Red	液体	0.2 mL	1.0 mL	-20℃.避光.	2 µL
A1524825F	标准品	粉末	4.2 mg	4.2 mg	-20℃.避光.	按需

使用方法

1. 试剂准备

(1) 将提取液，反应缓冲液，Amplex Red室温解冻，完全平衡至室温后再使用。

(2) 尿酸酶、过氧化物酶冰上解冻。

(3) 标准品配制：将标准品粉末用10 mL反应缓冲液完全溶解 (如不能完全溶解，可50℃水浴加热至完全溶解)，配制成终浓度2.5 mM的标准品溶液，然后分装成小管，-20℃避光保存，避免反复冻融。如果短时间内多次使用，也可暂放4℃保存。

2. 样品准备

(1) 细胞 (菌) 样本：离心收集细胞 (菌)后，建议每百万个细胞 (菌) 用100-200 μL提取液重悬，然后在4℃或冰浴等低温条件下进行超声破碎细胞 ( 200 W，超声2 S，间隔3 S，5 min)。4ºC, 12,000×g离心5分钟，取上清用于后续检测。

(2) 对于组织样品，每10 mg组织加入100-200 μL 提取液，在4℃或冰浴等低温条件下进行匀浆破碎。4ºC约12,000×g离心5分钟，取上清用于后续检测。

(3) 对于血清 (血浆) 样本：离心后可直接取上清检测，如超出检测上限，可用反应缓冲液稀释后再检测。

(4) 对于尿液样本：建议先用反应缓冲液稀释合适倍数后再检测，直接原液检测可能会导致结果偏低。

3. 显色工作液的配制

等反应缓冲液平衡至室温后，按照每个检测反应108 μL的体积配制显色工作液。根据待检测样品(包括标准品)的数量，配制适量的显色工作液。显色工作液的各个组份的具体配制方法参考下表。建议尽量现配现用，如果不能及时检测，请置于4℃或者冰浴避光保存且不要过夜，以免影响检测效果。

样本数	1	10	100	500
反应缓冲液	100 μL	1.0 mL	10 mL	50 mL
尿酸酶	4 μL	40 μL	400 μL	2.0 mL
过氧化物酶	2 μL	20 μL	200 μL	1.0 mL
Amplex Red	2 μL	20 μL	200 μL	1.0 mL

4. 样本测定

4.1 标准曲线绘制

(1) 吸光度检测：取25 μL尿酸标准品(2.5 mM) 添加100 μL ddH₂O稀释至0.5 mM (500 μM)，然后分别取500 μM的尿酸标准溶液0、1、2、5、10、20、50 μL加入96孔板中，并相应地用反应缓冲液补全至50 μL，此时，标准曲线的浓度分别为0、10、20、50、100、200、500 μM。

(2) 荧光法检测：取10 μL尿酸标准品(2.5 mM) 添加240 μL dd H₂O 稀释至0.1 mM (100 μM)，然后分别取100 μM的尿酸标准溶液0、1、2.5、5、10、25、50 μL加入96孔板中，并相应地用反应缓冲液补全至50 μL，此时，标准曲线的浓度分别为0、2、5、10、20、50、100 μM。

4.2 样品测定

(3) 取1-50 μL样品或稀释后的样品至96孔板样品孔中，并相应地加入检测缓冲液补足至50 µL。同时设置仅含检测缓冲液孔为空白对照孔。每孔加入显色工作液108 μL，混匀，37ºC避光反应30分钟。

(4) 反应结束后，如果是吸光度法检测，读取A₅₇₀；如果是荧光法检测，设置激发波长560 nm，发射波长590 nm进行荧光强度检测。

5. 结果展示

(1) 标准曲线 (仅供参考)。

(2) 建立标准曲线时，需要减去空白孔对照。标准曲线建立完毕后，可根据标准曲线计算出样品中的尿酸浓度（C），计算公示如下：

C(μM)=A×n

注：A为根据标准曲线计算出的尿酸浓度(μM)；n为样品稀释倍数。

注意事项

1. 显色工作液建议现用现配，且注意避光。

2. 检测尿液等液体样本时，建议先稀释合适倍数后看哪个稀释倍数信号值落在检测范围之内，然后选择该稀释倍数稀释样品后再检测，不建议直接用原液检测。

3. 本试剂盒仅用于科研用途，不得用于临床检测或其它用途。

4. 在实验过程中，请规范穿戴手套和实验服，避免皮肤直接接触试剂盒组分。

Product Introduction

Uric acid (UA) is the major metabolic end-product in birds and reptiles. In normal human urine, urea predominates with only a small amount of uric acid present. Furthermore, UA acts as a critical antioxidant capable of scavenging superoxide anions, hydroxyl radicals and other reactive species. Imbalance between endogenous UA production and excretion triggers a variety of diseases. For instance, elevated serum UA may lead to gout, renal impairment and arteriosclerosis, whereas reduced UA level is associated with pernicious anemia. Therefore, UA detection bears great significance in clinical diagnosis.

In this kit: Uricase catalyzes the oxidation of uric acid (UA) to produce allantoin, CO₂ and H₂O₂. In the presence of peroxidase, H₂O₂ further reacts with Amplex Red to form resorufin. The uric acid concentration in the sample is quantified by measuring the fluorescence intensity or absorbance of resorufin.

A1524825	Components	Appearance	100T	500T	Storage	volume per test
A1524825A	Uricase	liquid	0.4 mL	2.0 mL	-20℃.Store in the dark.	4 µL
A1524825B	Peroxidase	liquid	0.2 mL	1.0 mL	-20℃.Store in the dark.	2 µL
A1524825C	Extraction Buffer	liquid	20 mL	50 mL	-20℃.	As need
A1524825D	Reaction Buffer	liquid	30 mL	100 mL	-20℃.	100 µL
A1524825E	Amplex Red	liquid	0.2 mL	1.0 mL	-20℃.Store in the dark	2 µL
A1524825F	Standard	Powder	4.2 mg	4.2 mg	-20℃.Store in the dark	As need

Usage Method

1. Reagent Preparation

(1) Thaw Extraction Buffer, Reaction Buffer and Amplex Red at room temperature. Allow all reagents to fully equilibrate to room-temperature prior to use.

(2) Thaw uricase and peroxidase on ice.

(3) Standard preparation: Completely dissolve the standard powder with 10 mL Reaction Buffer. If full dissolution cannot be achieved, heat in a 50 °C water-bath until completely dissolved to prepare the standard solution with a final concentration of 2.5 mM. Aliquot into small tubes and store at −20 °C; avoid repeated freeze-thaw cycles. For frequent short-term use, temporary storage at 4 °C is acceptable.

2. Sample Preparation

(1) Cell (bacterial) samples: Collect cells (bacteria) by centrifugation. Resuspend every one million cells (bacteria) in 100-200 μL Extraction Buffer. Perform sonication for cell lysis under low-temperature conditions (4 °C or ice bath; 200 W, sonicate for 2 s, interval 3 s, total 5 min). Centrifuge at 12 000×g for 5 min at 4 °C, and collect the supernatant for subsequent detection.

(2) Tissue samples: Add 100-200 μL Extraction Buffer per 10 mg tissue. Homogenize for tissue lysis under low-temperature conditions (4 °C or ice bath). Centrifuge at approximately 12 000×g for 5 min at 4 °C, and collect the supernatant for subsequent detection.

(3) Serum (plasma) samples: The supernatant can be directly used for detection after centrifugation. If the value exceeds the upper detection limit, dilute the sample with Reaction Buffer prior to assay.

(4) Urine samples: It is recommended to dilute urine with Reaction Buffer to an appropriate fold-dilution before measurement. Direct testing of undiluted urine may result in underestimated results.

3. Preparation of Chromogenic Working Solution

After the Reaction Buffer has equilibrated to room temperature, prepare the chromogenic working solution at a volume of 108 μL per reaction. Prepare an appropriate amount according to the total number of samples to be tested (including standards). Refer to the table below for detailed formulation of each component of the chromogenic working solution. It is recommended to prepare fresh working solution immediately before use. If assay cannot be performed promptly, store the solution at 4 °C or on ice in the dark; do not store overnight to avoid compromising assay performance.

Number of sample	1	10	100	500
Reaction Buffer	100 μL	1.0 mL	10.0 mL	50 mL
Uricase	4 μL	40 μL	400 μL	2.0 mL
Peroxidase	2 μL	20 μL	200 μL	1.0 mL
Amplex Red	2 μL	20 μL	200 μL	1.0 mL

4. Sample Assay

4.1 Standard Curve Preparation

(1) Absorbance measurement: Pipette 25 μL uric acid standard (2.5 mM) and add 100 μL ddH₂O to dilute to 0.5 mM (500 μM). Transfer 0, 1, 2, 5, 10, 20, 50 μL of the 500 μM uric acid standard solution into separate wells of a 96-well plate, and make up the volume to 50 μL with reaction buffer accordingly. The resulting concentrations of the standard curve are 0, 10, 20, 50, 100, 200, 500 μM.

(2) Fluorescence measurement: Pipette 10 μL uric acid standard (2.5 mM) and add 240 μL ddH₂O to dilute to 0.1 mM (100 μM). Transfer 0, 1, 2.5, 5, 10, 25, 50 μL of the 100 μM uric acid standard solution into separate wells of a 96-well plate, and make up the volume to 50 μL with reaction buffer accordingly. The resulting concentrations of the standard curve are 0, 2, 5, 10, 20, 50, 100 μM.

4.2 Sample Measurement

(3) Transfer 1–50 μL sample or diluted sample into sample wells of a 96-well plate, and add assay buffer to bring the final volume to 50 μL. Set wells containing only assay buffer as blank controls. Add 108 μL chromogenic working solution to each well, mix well, and incubate at 37 °C in the dark for 30 min.

(4) Upon completion of incubation, read absorbance at A570 for absorbance assay. For fluorescence assay, detect fluorescence intensity with excitation wavelength at 560 nm and emission wavelength at 590 nm.

5. Calculation of Results

(1) Standard Curve (For reference only).

(2) When establishing the standard curve, the blank well control needs to be subtracted. After the standard curve is established, the uric acid concentration (C) in the sample can be calculated according to the standard curve, and the calculation formula is as follows:

C (μM) = A × n

Note: A represents the uric acid concentration (μM) calculated from the standard curve; n is the sample dilution factor.

Precautions

1. The chromogenic working solution should preferably be prepared freshly before use and protected from light.

2. When testing liquid samples such as urine, dilute the sample by appropriate fold-dilutions first to identify which dilution yields signals within the assay detection range. Perform the assay using this optimized dilution. Testing undiluted raw sample is not recommended.

3. This kit is for research-only use. It shall not be applied for clinical diagnosis or any other purposes.

4. During the experiment, wear gloves and lab coats as standard laboratory practice to avoid direct skin contact with kit components.

规格

规格或纯度

BioReagent, 比色法, 适用于分析, 标曲法

英文名称

Uric Acid (UA) Content Assay Kit (Fluorometric/Colorimetric, Microplate)

应用

细胞代谢

检测范围

荧光法, 1 μM-100 μM; 吸光度法, 5 μM-500 μM

检测方法

荧光法/吸光度法, 微量法

定量&半定量

定量

检测仪器

酶标仪

检测波长

Absorption method, 570 nm; fluorescence method, excitation wavelength 560 nm, emission wavelength 590 nm

敏感性

Light-sensitive

检测时长

30 min

样本体积

50 μL

储存与运输

储存条件

-20°C储存

运输条件

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1年。

组分与储存

A1524825	组件	物理外观	100T	500T	储存	单次测试使用量
A1524825A	尿酸酶	液体	0.4 mL	2.0 mL	-20℃.避光	4 µL
A1524825B	过氧化物酶	液体	0.2 mL	1.0 mL	-20℃.避光	2 µL
A1524825C	提取液	液体	20 mL	50 mL	-20℃.	按需
A1524825D	反应缓冲液	液体	30 mL	100 mL	-20℃.	100 µL
A1524825E	Amplex Red	液体	0.2 mL	1.0 mL	-20℃.避光	2 µL
A1524825F	标准品	粉末	4.2 mg	4.2 mg	-20℃.避光	按需

技术文档

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高级数据

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溶液计算器

摩尔浓度计算器

计算溶液所需的质量、体积或浓度。

质量

浓度

体积

分子量

g/mol

稀释计算器

计算配制储备溶液所需的稀释度。

浓度1（C1）

体积 1 (V1)

浓度 2 (C2)

体积 2 (V2)

复溶计算器

复溶计算器允许您快速计算试剂的体积，以复溶小瓶。只需输入试剂的质量和目标浓度，计算器将确定其余部分。

体积（添加到小瓶中）

质量（小瓶）

所需的复溶浓度

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尿酸 (UA) 含量检测试剂盒 (荧光法/吸光度法, 微量法)