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。
血磷主要指血中以无机磷盐的形式存在的无机磷，血浆中钙、磷浓度对骨盐的形成和骨的钙化等 具有重要影响，两者含量的相对稳定依赖于钙、磷的吸收与排泄和钙化与脱钙两种代谢的相对平衡，血磷含量可作为多种疾病的检测指标之一，在医学检测领域具有重要应用。
检测原理：血清样本经处理去除有机磷后，无机磷能够与钼酸铵反应生成磷钼酸，被硫酸亚铁还原后呈蓝色，通过测定620nm吸光度，计算血液中磷含量。
注意：正式检测前，建议选择2-3个预期差异较大的样本进行预实验。
自备仪器和试剂
操作步骤
1、试剂准备
| 试剂名称 | 试剂准备 | 备注 |
| Reagent Ⅰ | 即用型，使用前平衡到室温 | 4℃保存；有毒且有刺激性气味，建议在通风橱进行实验。 |
| Reagent Ⅱ | 即用型，使用前平衡到室温 | 4℃避光保存 |
| Working Reagent Ⅲ | 临用前配制，加入11mL去离子水充分溶解后使用，再加入Reagent Ⅱ，充分混合。 | 仅当天使用 |
| Standard | 即用型，使用前平衡到室温 | 4℃保存 |
2、样本制备
血清样品：吸取50μL血清，加950μL Reagent Ⅰ，混匀后室温8,000rpm，离心10min，取上清液，待测。
3、实验步骤
3.1 酶标仪或可见分光光度计预热30min，调节波长到620nm。可见分光光度计用去离子水调零。
3.2 操作表（下述操作在微量玻璃比色皿/96孔板中进行）：
| 试剂（μL） | 空白孔 | 标准孔 | 测定孔 |
| 样本上清 | 0 | 0 | 50 |
| 去离子水 | 50 | 0 | 0 |
| Standard | 0 | 50 | 0 |
| Reagent Ⅰ | 50 | 50 | 50 |
| Working Reagent Ⅲ | 100 | 100 | 100 |
3.3 混匀后室温静置10min，于620nm测定吸光度，记为A空白、A标准、A测定，计算ΔA测=A测定-A空白、ΔA标=A标准;-A空白。
注意：空白孔和标准孔只需测定1次。实验之前建议选择2-3个预期差异大的样本做预实验。如果ΔA测过小可适当加大样本量。如果ΔA测大于1.0，样本可用Reagent Ⅰ进一步稀释，计算结果乘以稀释倍数，或减少提取用样本量。
4、结果计算
注意：我们为您提供的计算公式，包括推导过程计算公式和简洁计算公式。两者完全相等。建议以加粗的简洁计算公式为最终计算公式。
血磷浓度计算：
血磷含量 (mmol/dL)=(CStandard×ΔA测÷ΔA标)×样品稀释倍数=2×ΔA测÷ΔA标
参数说明：
CStandard：0.1 mmol/dL；
样品稀释倍数：(50μL 血清+950μL Reagent Ⅰ)÷50μL 血清=20。
5、结果展示
图1：山羊血磷含量
注意事项
1、Working Reagent Ⅲ需临用前配制，限当天使用。
2、测定过程中，应尽量避免溶血，因为红细胞中有机磷酯进入血清后可被酶水解而使得血清无机磷含量增高。
3、本产品仅供科学研究使用，不适用于临床诊断。为了您的安全和健康，请穿实验服并戴一次性手套操作。
Blood phosphorus mainly refers to inorganic phosphorus existing in the blood in the form of inorganic phosphate salts. The concentrations of calcium and phosphorus in plasma significantly influence bone salt formation and bone calcification. The relative stability of their contents depends on the balance between calcium/phosphorus absorption and excretion, and the equilibrium between calcification and decalcification. Blood phosphorus levels can serve as one of the detection indicators for various diseases, holding significant application value in the field of medical testing.
Detection Principle
After processing the serum sample to remove organic phosphorus, inorganic phosphorus reacts with ammonium molybdate to form phosphomolybdic acid. Upon reduction by ferrous sulfate, it turns blue. The phosphorus content in blood is calculated by measuring the absorbance at 620 nm.
Note: Before formal testing, it is recommended to perform a preliminary experiment with 2-3 samples expected to show significant differences.
Reagents, consumables and Equipments not provided
Microplate reader or visible spectrophotometer (capable of measuring absorbance at 620 nm)
96-well plate or micro-volume glass cuvette, Adjustable pipettes and tips
Centrifuge
Deionized water
Procedure
1. Reagent Preparation
| Reagent Name | Preparation | Notes |
| Reagent Ⅰ | Ready-to-use. Equilibrate to room temperature before use. | Store at 4°C. Toxic and irritating odor; perform experiment in a fume hood. |
| Reagent Ⅱ | Ready-to-use. Equilibrate to room temperature before use. | Store at 4°C protected from light. |
| Working Reagent Ⅲ | Prepare immediately before use. Dissolve Reagent Ⅲ thoroughly in 11 mL deionized water, then add Reagent Ⅱ and mix well. | Use on the same day. |
| Standard | Ready-to-use. Equilibrate to room temperature before use. | Store at 4°C. |
2. Sample Preparation
Serum Sample: Pipette 50 µL of serum, add 950 µL of Reagent Ⅰ, mix well, centrifuge at 8,000 rpm at room temperature for 10 minutes. Collect the supernatant for testing.
3. Experimental Steps
3.1 Preheat the microplate reader or visible spectrophotometer for 30 minutes and set the wavelength to 620 nm. Zero the visible spectrophotometer with deionized water.
3.2 Operation Table (perform in micro-volume glass cuvette / 96-well plate):
| Reagent (µL) | Blank Well | Standard Well | Test Well |
| Sample Supernatant | 0 | 0 | 50 |
| Deionized Water | 50 | 0 | 0 |
| Standard | 0 | 50 | 0 |
| Reagent Ⅰ | 50 | 50 | 50 |
| Working Reagent Ⅲ | 100 | 100 | 100 |
3.3 Mix well and incubate at room temperature for 10 minutes. Measure the absorbance at 620 nm, recorded as A<sub>Blank, A<sub>Standard, A<sub>Test. Calculate ΔA<sub>Test = A<sub>Test</sub> - A<sub>Blank and ΔA<sub>Standard = A<sub>Standard</sub> - A<sub>Blank</sub>.
Note: The blank and standard wells only need to be measured once. It is recommended to perform a preliminary experiment with 2-3 samples showing expected large differences before formal testing. If ΔA<sub>Test</sub> is too small, appropriately increase the sample amount. If ΔA<sub>Test</sub> is greater than 1.0, further dilute the sample with Reagent Ⅰ and multiply the result by the dilution factor, or reduce the amount of sample used for extraction.
4. Calculation of Results
Note: We provide two formula sets: the derived calculation formulas and the simplified formulas. They are completely equivalent. The simplified formulas are recommended as the final calculation formulas.
Blood Phosphorus Concentration Calculation:
Blood Phosphorus Content (mmol/dL) = (C;Standard × ΔATest ÷ ΔAStandard) × Sample Dilution Factor = 2 × ΔATest ÷ ΔA;Standard
Parameter Description:
C<sub>Standard</sub>: 0.1 mmol/dL;
Sample Dilution Factor: (50 µL serum + 950 µL Reagent Ⅰ) ÷ 50 µL serum = 20.
5. Example Results
Figure 1: Goat Blood Phosphorus Content
Notes
Working Reagent Ⅲ must be prepared immediately before use and is limited to use on the same day.
Hemolysis should be avoided during the assay procedure, as organic phosphoesters from red blood cells entering the serum can be hydrolyzed by enzymes, leading to increased serum inorganic phosphorus levels.
This product is for scientific research use only and is not intended for clinical diagnosis. For your safety and health, please wear a lab coat and disposable gloves during operation.
| B1507967 | Component | 96T | Storage |
| B1507967A | Reagent Ⅰ | 65 mL×2 | 2-8℃ |
| B1507967B | Reagent Ⅱ | 1.6 mL | 2-8℃. Store in the dark. |
| B1507967C | Reagent Ⅲ | 1 EA | 2-8℃ |
| B1507967D | Standard | 2 mL | 2-8℃ |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0131663 |
分析证书 | B1507967 |
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