BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
尿中出现蛋白称为蛋白尿，即尿蛋白，正常尿中有极微量蛋白，正常情况下尿蛋白定性结果应为阴性，当出现肾脏病变或损伤等情况时，多表现为尿中蛋白质含量持续增多，尿蛋白定性结果为阳性， 其含量变化常作为肾病诊断以及判定损伤程度的重要依据。
检测原理：丽春红S染料与尿液样本中的蛋白质结合形成不溶性的蛋白质-染料复合物。经离心沉淀后，弃去上清，将沉淀物溶解于碱性缓冲液中。此时，复合物解离，染料恢复其颜色特性。使用分光光度计在560 nm波长下检测吸光度，其值与蛋白质浓度成正比，通过与标准曲线对比即可计算出样本中的蛋白含量。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
| H1506744 | Component | 100T | Storage |
| H1506744A | 磺基水杨酸溶液 | 100 mL | RT. Store in the dark. |
| H1506744B | 丽春红S试剂 (10×) | 10 mL | RT. Store in the dark. |
| H1506744C | Alkaline Buffer | 200 mL | RT. |
| H1506744D | 蛋白标准 (BSA) | 20 mg | RT. |
| H1506744E | 蛋白标准配制液 | 1.5 mL | RT. |
自备仪器和试剂
1、蒸馏水
实验流程
1、配制1×丽春红S工作液
取适量丽春红S试剂 (10×)，按1:9比例加入蒸馏水进行稀释（例如，1 mL 10×试剂 + 9 mL 水），混匀。室温避光保存，可稳定2-3个月。
2、配制蛋白标准品原液 (20 mg/mL)
取1ml蛋白标准配制液加入到蛋白标准 (BSA) 中，充分溶解后配制成 20mg/mL 的蛋白标准溶液，配制后可立即使用，溶解后的蛋白标准溶液应-20℃保存。
3、配制蛋白标准品稀释液
取适量20mg/mL蛋白标准，稀释至浓度分别为0、200、400、600、800、1000、1200、1600μg/mL或所需浓度。请注意：为保持基质一致，用于稀释标准品的溶液应与待测尿样的基质相同。例如待测蛋白溶解于蔗糖中，亦取20mg/mL蛋白标准溶解于蔗糖中，一般也可以用0.9%NaCl或PBS作为溶解BSA稀释液。稀释后的蛋白标准也应-20℃长期保存。
4、制作蛋白标准曲线
各取100μL上述稀释度的蛋白标准， 与测定样本操作相同（见第6步）。”0”号管空白调零，比色杯光径1cm，560nm比色，制成标准曲线。
5、半定量预实验
取3-5 mL新鲜尿液于小试管中。沿管壁缓缓滴加2-3滴磺基水杨酸溶液，使其在尿液表面形成界面。立即在黑色背景下观察界面现象，根据下表判断尿蛋白的大致浓度范围：
| 结果判定 | 现象描述 | 预估蛋白浓度 |
| 阴性 (-) | 不显浑浊，清晰透明 | < 0.05 g/L |
| 可疑 (±) | 轻微浑浊，隐约可见 | 0.05 - 0.2 g/L |
| (+) | 明显白色浑浊，无颗粒 | ~ 0.3 g/L |
| (2+) | 稀薄乳样浑浊，出现颗粒 | ~ 1 g/L |
| (3+) | 乳浊，有絮片状沉淀 | ~ 3 g/L |
| (4+) | 絮片状沉淀，凝固成块 | > 5 g/L |
6、定量检测
6.1 确定样本用量
根据半定量预实验的结果，参照下表选择合适的样本体积进行检测：
| 预估蛋白浓度 | 待测样本体积 | 稀释倍数 | |
| < 1 g/L | 100 μL | 0 μL | 1 |
| 1 - 3 g/L | 50 μL | 50 μL | 2 |
| > 3 g/L | 10 μL | 90 μL | 10 |
6.2 沉淀反应
在离心管中依次加入上表确定的待测样本、蒸馏水，再加入1 mL 1×丽春红S工作液，涡旋混匀。3500 g离心10min。
6.3 清洗与溶解
离心后将上清缓缓倒出后，倒置于滤纸上数分钟，用滤纸条吸附管壁上多余的试剂（勿触及管低沉淀），再加入2ml Alkaline Buffer至沉淀中，充分混合使沉淀溶解。
6.4 比色测定
用分光光度计，以蒸馏水调零，在560 nm波长下（540-595 nm亦可）测定各标准品和样本的吸光度值。
7、结果计算
7.1 以蛋白标准品稀释液的浓度（μg/mL）为横坐标，以其对应的吸光度值为纵坐标，绘制标准曲线，并获得回归方程。
7.2 将样本的吸光度值代入回归方程，计算出样本的测定浓度（C，单位为 μg/mL）。
7.3 根据测定时样本的稀释倍数，计算原尿液的蛋白浓度：
原尿液蛋白浓度 (g/L) = [C (μg/mL) × 稀释倍数] / 1000
参考区间（患有肾病的患者）：46.5±18.1 mg/L
注意事项
1、蛋白标准 (BSA) 粉末溶解于蛋白标准配制液后，即获得蛋白标准原液，该原液中含有防腐剂，不影响后续检测，该蛋白标准原液-20℃长期保存。
2、待测蛋白溶解于什么样的稀释液中，蛋白标准也宜溶解于什么样的稀释液中，否者待测蛋白与蛋白标准中所含非蛋白成分不一致，有可能导致测定不准确。
3、待测样本蛋白含量＜0.1g/L时，可用1ml样本加0.1ml丽春红S试剂 (10×)，混匀，余下操作同上。
4、该试剂盒也可以用酶标仪检测。
5、为了您的安全和健康，请穿实验服并戴一次性手套操作。
6、试剂开封后请尽快使用，以防影响后续实验效果。
The presence of protein in urine is termed proteinuria. While normal urine contains minimal trace amounts of protein, qualitative tests under healthy conditions should yield a negative result. Persistent elevation of urinary protein levels, indicated by a positive qualitative test, often signifies renal pathology or impairment. Consequently, changes in urinary protein concentration are a critical parameter for diagnosing kidney disease and assessing the extent of damage.
Detection Principle
Ponceau S dye binds to proteins in the urine sample, forming an insoluble protein-dye complex. After centrifugation, the supernatant is discarded, and the pellet is dissolved in an alkaline buffer. This dissociates the complex, restoring the dye's colorimetric properties. The absorbance is measured at 560 nm using a spectrophotometer. The absorbance value is proportional to the protein concentration, allowing for the calculation of protein content in the sample by comparison with a standard curve. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
| H1506744 | Component | 100T | Storage |
| H1506744A | Sulfosalicylic Acid Solution | 100 mL | RT. Store in the dark. |
| H1506744B | Ponceau S Reagent (10X) | 10 mL | RT. Store in the dark. |
| H1506744C | Alkaline Buffer | 200 mL | RT. |
| H1506744D | Protein Standard (BSA) | 20 mg | RT. |
| H1506744E | Protein Standard Diluent | 1.5 mL | RT. |
Materials Required but Not Provided
Distilled water
Centrifuge tubes, test tubes, cuvettes, spectrophotometer
Experimental Procedure
1. Preparation of 1X Ponceau S Working Solution
Dilute an appropriate amount of the Ponceau S Reagent (10X) with distilled water at a 1:9 ratio (e.g., 1 mL of 10X Reagent + 9 mL water). Mix well. This working solution can be stored at room temperature protected from light and remains stable for 2-3 months.
2. Preparation of Protein Standard Stock Solution (20 mg/mL)
Add 1 mL of Protein Standard Diluent to the vial containing the Protein Standard (BSA). Dissolve completely to prepare a 20 mg/mL protein standard stock solution. The dissolved stock solution can be used immediately and should be stored at -20°C for long-term preservation.
3. Preparation of Diluted Protein Standard Solutions
Dilute the 20 mg/mL Protein Standard Stock to concentrations such as 0, 200, 400, 600, 800, 1000, 1200, 1600 μg/mL, or other desired concentrations.
Note: To maintain matrix consistency, the solution used for diluting the standards should ideally match the matrix of the test urine samples. For instance, if the target protein is dissolved in sucrose, the 20 mg/mL standard should also be diluted in sucrose. Generally, 0.9% NaCl or PBS can also be used to dilute the BSA standard. Diluted protein standards should also be stored at -20°C.
IV. Generating the Protein Standard Curve
Take 100 μL of each diluted protein standard and process them identically to the test samples (see Step 6). Use the "0" concentration standard as the blank to zero the instrument. Measure the absorbance at 560 nm using a cuvette with a 1 cm path length. Plot the standard curve using concentration vs. absorbance.
4. Semi-Quantitative Preliminary Test
4.1 Place 3-5 mL of fresh urine in a small test tube.
4.2 Slowly add 2-3 drops of Sulfosalicylic Acid Solution down the inner wall of the tube to form a layer on top of the urine.
4.3 Immediately observe the interface against a black background. Estimate the approximate protein concentration range based on the following table:
| Result Interpretation | Phenomenon Description | Estimated Protein Concentration |
| Negative (-) | No turbidity, clear and transparent | < 0.05 g/L |
| Suspicious (±) | Slight turbidity, faintly visible | 0.05 - 0.2 g/L |
| (+) | Distinct white turbidity, no granules | ~ 0.3 g/L |
| (2+) | Thin, milky turbidity with appearing granules | ~ 1 g/L |
| (3+) | Milky and opaque, with flocculent precipitates | ~ 3 g/L |
| (4+) | Flocculent precipitates, coagulated into clumps | > 5 g/L |
6. Quantitative Detection
6.1 Determining Sample Volume
Based on the result of the semi-quantitative test, select an appropriate sample volume for detection according to the table below:
| Estimated Protein Concentration | Sample Volume | Dilution Factor | |
| < 1 g/L | 100 μL | 0 μL | 1 |
| 1 - 3 g/L | 50 μL | 50 μL | 2 |
| > 3 g/L | 10 μL | 90 μL | 10 |
6.2 Precipitation Reaction
Add the determined sample volume and distilled water (if any) to a centrifuge tube. Add 1 mL of 1X Ponceau S Working Solution. Vortex to mix thoroughly. Centrifuge at 3500 × g for 10 minutes.
6.3 Washing and Dissolution
After centrifugation, carefully decant the supernatant. Invert the tube on absorbent paper for several minutes to drain. Use a filter paper strip to wipe away any excess reagent from the tube walls (avoid touching the pellet). Add 2 mL of Alkaline Buffer to the pellet and mix thoroughly to dissolve the precipitate.
6.4 Colorimetric Measurement
Using a spectrophotometer zeroed with distilled water, measure the absorbance of all standard and sample solutions at 560 nm (540-595 nm is also acceptable).
7. Calculation of Results
7.1 Plot the standard curve using the concentrations (μg/mL) of the diluted protein standards as the x-axis and their corresponding absorbance values as the y-axis. Obtain the regression equation.
7.2 Substitute the sample's absorbance value into the regression equation to calculate the measured concentration (C, in μg/mL).
7.3 Calculate the protein concentration in the original urine sample based on the dilution factor used during the assay:
Original Urine Protein Concentration (g/L) = [C (μg/mL) × Dilution Factor] / 1000
Reference Interval (in patients with kidney disease): 46.5 ± 18.1 mg/L
Precautions
The Protein Standard (BSA) powder dissolved in the Protein Standard Diluent yields the protein standard stock solution. This stock contains preservatives which do not interfere with the assay. This stock solution can be stored long-term at -20°C.
The protein standards should ideally be dissolved/diluted in the same type of solution as the protein in the test samples. Inconsistent non-protein components between the sample and standard matrices may lead to inaccurate results.
If the estimated protein concentration in the test sample is < 0.1 g/L, use 1 mL of sample mixed directly with 0.1 mL of Ponceau S Reagent (10X). Mix well and proceed with the remaining steps.
This kit is also compatible with detection using a microplate reader.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use reagents promptly after opening to prevent potential effects on subsequent experimental results.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131311 |
分析证书 | H1506744 |
危险品化学品经营许可证（带存储）