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。
血清中的无机磷（Inorganic Phosphorus）主要由H₂PO₄⁻和HPO₄²⁻两种磷酸根阴离子组成，上述阴离子在不同的pH环境下能快速相互转换。在pH7.4血清中两种磷酸根阴离子浓度比例为1:4，在酸中毒环境下二者浓度约为1:1，在碱中毒环境下二者浓度比例为1:9，在pH4.5尿液中浓度比例为100:1。WHO推荐的常规检测方法为比色法，我国卫生部临检中心推荐的常规方法为硫酸亚铁钼蓝比色法和米吐尔钼蓝比色法。
检测原理：先经硫酸亚铁提纯蛋白，利用无机磷与钼酸铵结合生成磷钼酸铵，后者被硫酸亚铁还原成蓝紫色的复合物，通过分光光度计检测640nm处吸光度，根据公式计算出无机磷含量。本试剂盒仅用于科研领域，不宜用于临床诊断或其他用途。
注：整套试剂盒未拆封时于2-8℃避光保存，自生产之日起有效期 1 年；拆封后各组分稳定性不同，部分组分可转至RT保存（有效期仍为 1 年），其余需继续2-8℃保存。请严格按照组分表标注温度分类存放，保障试剂性能。 |
自备仪器和试剂
1、去离子水
2、电子天平、离心管或试管、离心机、比色杯、分光光度计
实验流程
1、制备样品
1.1 血浆、血清样品
取0.1ml待测血浆、血清样品，加入2.4ml Pi Assay Buffer，充分混匀，室温静置10min，3000g离心10min，取上清，-20℃冻存，用于Pi的检测。
1.2 尿液样品
取适量的待测尿液，用50%的盐酸调pH至6.0，用蒸馏水做1:10~20稀释。取0.1ml稀释后的尿液样品，加入2.4ml Pi Assay Buffer，充分混匀，室温静置10min，3000g离心10min，取上清，-20℃冻存，用于Pi的检测。
1.3 细胞或组织样品
取恰当细胞或组织进行匀浆，低速离心取上清，取0.1ml匀浆样品，加入2.4ml Pi Assay Buffer，充分混匀，室温静置10min，3000g离心10min，取上清，-20℃冻存，用于Pi的检测。
1.4 高浓度样品
如果样品中含有较高浓度的Pi，可以使用去离子水稀释。
注：样品准备完毕后可以用BCA蛋白浓度测定试剂盒测定蛋白浓度，以便于后续计算单位蛋白重量组织或细胞内的Pi含量。推荐使用阿拉丁B665595 BCA蛋白定量试剂盒或R1491648 即用型BCA蛋白定量试剂盒。
2、制备磷标准工作液
取适量的磷标准 (1mg/mL)，按磷标准 (1mg/mL)：标准品稀释液=1:24的比例稀释，即获得磷标准 (1.292mmol/L)；取0.1ml磷标准 (1.292mmol/L)，加入2.4ml Pi Assay Buffer，充分混匀，室温静置10min，3000g离心10min，取上清，即为磷标准工作液，-20℃冻存，用于Pi的检测。
3、配制钼酸铵显色液
将钼酸铵粉末和钼酸铵稀释液按0.44g：10ml的比例混合溶解，即可使用。钼酸铵显色液不宜长期保存，一般配制后4℃保存，2周有效。
4、Pi加样
按照下表设置空白管、标准管、测定管，溶液应按照顺序依次加入，并注意避免产生气泡，按下表操作。如果样品中的Pi含量过高，可以减少样品用量或适当稀释后再进行测定，样品的检测最好能设置2平行管，求平均值。
| 加入物 (ml) | 空白管 | 标准管 | 测定管 |
| Pi Assay Buffer | 2 | — | — |
| 处理后的磷标准工作液 | — | 2 | — |
| 处理后的待测上清液 | — | — | 2 |
| 钼酸铵显色液 | 0.25 | 0.25 | 0.25 |
5、Pi测定
混匀，室温静置15min，以空白管调零，比色杯光径1cm，分光光度计640nm处检测标准管、测定管的吸光度（即为A标准，A测定）。
6、结果计算
血清、血浆中无机磷计算公式：磷 (mmol/L)=(A测定/A标准)×1.292
尿液中无机磷计算公式：磷 (mmol/d)=(A测定/A标准)×1.292×N×24h尿量(L)
组织中磷计算公式：磷 (mmol/mg)=(A测定/A标准)×1.292/待测样品蛋白浓度(mg/L)
参数说明：
A测定：待测管的吸光度
A标准：标准管的吸光度
N：尿液稀释倍数
单位换算：1 mg/dL=10 mg/L=1 mmol/L×0.323
参考区间：
| 样本 | 血清磷浓度 |
| 健康成年人 | 0.96~1.62mmol/L (3~5mg/dl) |
| 儿童 | 1.45~2.1mmol/L (4.5~6.5mg/dl) |
注意事项
1、溶血样本对检测有干扰，尽量避免采用溶血样本。
2、本法能够用于自动生化分析仪终点检测法。
3、如果样品浓度过高，应用蒸馏水稀释后重测，结果乘以稀释倍数。
4、本法测定的线性范围为0.01~0.2mg/ml，检测下限为0.005，检测上限为1。
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、组分I1506776C和 I1506776E具有腐蚀性，使用时请佩戴手套，做好个人防护。
附录：标准曲线制作：在室温条件下按说明书操作，用分光光度计对系列磷标准 (1/100、1/40、1/25、1/15、1/10、1/8 mg/ml)进行吸光度的测定，其标准曲线如下(仅供参考)：
Inorganic phosphorus in serum primarily consists of two phosphate anions, H₂PO₄⁻ and HPO₄²⁻, which can rapidly interconvert depending on the pH environment. In serum at pH 7.4, the concentration ratio of these two anions is approximately 1:4. This ratio shifts to about 1:1 in acidosis and 1:9 in alkalosis. In urine at pH 4.5, the ratio is approximately 100:1. The colorimetric method is the routine assay recommended by the WHO. The Chinese Ministry of Health Clinical Test Center recommends the ferrous sulfate molybdenum blue method and the metol molybdenum blue method as routine procedures.
Detection Principle
Proteins are first precipitated and purified using ferrous sulfate. Inorganic phosphorus reacts with ammonium molybdate to form ammonium phosphomolybdate, which is then reduced by ferrous sulfate to form a blue-purple complex. The absorbance of this complex is measured at 640 nm using a spectrophotometer, and the inorganic phosphorus content is calculated based on a formula. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Materials Required but Not Provided
1. Deionized water
2. Electronic balance, centrifuge tubes or test tubes, centrifuge, cuvettes, spectrophotometer
Note: The entire kit should be stored at 2-8℃ in a dark place before opening. It is valid for 1 year from the date of production. After opening, the stability of different components varies. Some components can be stored at RT (the validity period remains 1 year), while the rest need to be stored at 2-8℃. Please store each component according to the temperature indicated in the component table to ensure the performance of the reagents. |
Experimental Procedure
1. Sample Preparation
1.1 Plasma/Serum Samples
Add 0.1 ml of the plasma or serum sample to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.
1.2 Urine Samples
Adjust the pH of an appropriate amount of urine to 6.0 using 50% hydrochloric acid. Dilute 1:10 to 1:20 with distilled water. Add 0.1 ml of the diluted urine sample to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.
1.3 Cell or Tissue Samples
Homogenize an appropriate amount of cells or tissue. Centrifuge at low speed to collect the supernatant. Add 0.1 ml of the homogenate supernatant to 2.4 ml of Pi Assay Buffer. Mix thoroughly and let stand at room temperature for 10 minutes. Centrifuge at 3000 × g for 10 minutes. Collect the supernatant and store at -20°C for Pi detection.
1.4 High Concentration Samples
If the sample contains a high concentration of Pi, it can be diluted with deionized water.
Note: After sample preparation, the protein concentration can be determined using a BCA protein assay kit to facilitate subsequent calculation of Pi content per unit protein weight in tissues or cells. The Aladdin B665595 BCA Protein Quantification Kit or R1491648 Ready-to-Use BCA Protein Quantification Kit is recommended.
2. Preparation of Phosphorus Standard Working Solution
Dilute an appropriate amount of the Phosphorus Standard (1 mg/mL) with Standard Diluent at a ratio of 1:24 (Phosphorus Standard : Standard Diluent) to obtain a Phosphorus Standard (1.292 mmol/L). Add 0.1 ml of this Phosphorus Standard (1.292 mmol/L) to 2.4 ml of Pi Assay Buffer. Mix thoroughly, let stand at room temperature for 10 minutes, and centrifuge at 3000 × g for 10 minutes. Collect the supernatant; this is the Phosphorus Standard Working Solution. Store at -20°C for Pi detection.
3. Preparation of Ammonium Molybdate Chromogenic Solution
Dissolve the Ammonium Molybdate Powder in the Ammonium Molybdate Diluent at a ratio of 0.44 g powder per 10 ml diluent. Mix until dissolved. The prepared Ammonium Molybdate Chromogenic Solution should not be stored long-term; it is generally stable for 2 weeks when stored at 4°C after preparation.
4. Pi Assay Setup
Set up Blank, Standard, and Test tubes according to the table below. Add solutions in the order specified, avoiding bubble formation. If the Pi content in the sample is too high, reduce the sample volume or dilute appropriately before assay. It is best to set up duplicate tubes for samples and use the average value.
| Reagent (ml) | Blank Tube | Standard Tube | Test Tube |
| Pi Assay Buffer | 2 | — | — |
| Treated Std Working Solition | — | 2 | — |
| Treated Sample Supernatant | — | — | 2 |
| Ammonium Molybdate Chromogenic Solution | 0.25 | 0.25 | 0.25 |
5. Pi Measurement
Mix well and incubate at room temperature for 15 minutes. Using the Blank tube to zero the spectrophotometer, measure the absorbance of the Standard tube and Test tubes at 640 nm using a cuvette with a 1 cm light path. Record the values as Astandard and Atest .
6. Calculation of Results
Serum/Plasma Inorganic Phosphorus:
Phosphorus (mmol/L) = (Atest / Astandard) × 1.292
Urine Inorganic Phosphorus:
Phosphorus (mmol/d) = (Atest / Astandard) × 1.292 × N × 24-hour urine volume (L)
Tissue Inorganic Phosphorus:
Phosphorus (mmol/mg prot) = Atest / Astandard) × 1.292 / Protein concentration of test sample (mg/L)
Parameter Description:
Atest: Absorbance of the test tube
Astandard: Absorbance of the standard tube
N: Urine dilution factor
Unit Conversion: 1 mg/dL = 10 mg/L = (1 mmol/L) × 0.323
Reference Interval:
| Sample Type | Serum Phosphorus Concentration |
| Healthy Adults | 0.96~1.62mmol/L (3~5mg/dl) |
| Children | 1.45~2.1mmol/L (4.5~6.5mg/dl) |
Precautions
Hemolyzed samples interfere with the assay; avoid using them if possible.
This method can be adapted for automated biochemical analyzers using an endpoint detection method.
If the sample concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.
The linear range of this assay is 0.01 ~ 0.2 mg/ml. The lower limit of detection is 0.005, and the upper limit is 1.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Components I1506776C and I1506776E are corrosive; please wear gloves and take appropriate personal protective measures when handling them.
Appendix: Standard Curve Preparation
When operated at room temperature according to the instructions, measure the absorbance of a series of phosphorus standards (1/100, 1/40, 1/25, 1/15, 1/10, 1/8 mg/ml) using a spectrophotometer. The resulting standard curve is shown below (for reference only):
| 组分货号 | 组分名称 | 物理外观 | 50T | 100T | 储存 | 单次测试使用量 |
| I1506776A | 磷标准 (1mg/mL) | 无色透明液体 | 1 mL | 2 mL | 2-8℃ | 按需求使用 |
| I1506776B | 标准品稀释液 | 无色透明液体 | 25 mL | 50 mL | 室温，避光 | 按需求使用 |
| I1506776C | Pi Assay Buffer | 无色透明液体 | 250 mL | 500 mL | 2-8℃，避光 | 每次反应需使用2 mL |
| I1506776D | 钼酸铵粉末 | 白色粉末 | 0.66 g | 1.32 g | 室温 | 按需求使用 |
| I1506776E | 钼酸铵稀释液 | 无色透明液体 | 15 mL | 30 mL | 室温 | 每次反应需使用0.25 mL |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ25F1230308 |
分析证书 | I1506776 |
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