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总蛋白（Total Protein,TP）由白蛋白和球蛋白组成，检测白蛋白的方法有双缩脲法、色氨酸法、染料结合法，其中检测白蛋白的染料结合法可采用溴甲酚绿或溴甲酚紫染料结合，上述染料对白蛋白具有高度的亲和力，通常监测染料与白蛋白结合的初速率，该速率与样品中白蛋白浓度成正比。
检测原理：在酸性环境下，白蛋白分子带正电荷，与带负电荷的溴甲酚绿（Bromocresol green，BCG）结合生成蓝绿色复合物，在628nm处有吸收波，该复合物的吸光度与白蛋白浓度成正比，与同样处理的白蛋白标准比较，求得待测样品中白蛋白浓度，多用于人或动物血清、血浆、组织等样本中的蛋白含量测定，该法操作简单、方法特异，既可手工操作，又可采用自动分析仪检测。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
| A1505873 | Component | 100T | Storage |
| A1505873A | BCG试剂 | 25 mL | 2-8℃. Store in the dark. |
| A1505873B | 白蛋白标准 | 20 mg | RT |
| A1505873C | 白蛋白标准配制液 | 2 mL | RT |
| A1505873D | BCG空白试剂 | 10 mL | RT |
自备仪器和试剂
离心管、小试管、酶标仪、96孔板
实验流程
1、白蛋白标准溶液配制
取0.5ml白蛋白标准配制液或稀释液加入到白蛋白标准 (20mg)，充分溶解后配制成40mg/ml白蛋白标准溶液，配制后可立即使用，溶解后的白蛋白标准溶液应-20℃保存。
特别提示：待测蛋白溶解于什么样的稀释液中，白蛋白标准也宜溶解于什么样的稀释液中，例如待测蛋白溶解于蔗糖，亦取白蛋白标准溶解于蔗糖，一般也可以用0.9%NaCl或PBS作为溶解白蛋白标准品的稀释液。
2、样本处理
血清、血浆样本：直接取5μl检测。
组织样本：按组织质量(g)：生理盐水(ml)=1：9比例，加入9倍体积的生理盐水或PBS，冰浴下匀浆后，2500g离心10min，取5μl上清待检。
3、白蛋白加样
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。如果样品浓度过高，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平行孔。
| 加入物 (μl) | 空白孔 | 标准孔 | 测定孔 |
| 白蛋白标准配制液 | 5 | − | − |
| 白蛋白标准溶液 (40mg/ml) | − | 5 | − |
| 待检样品 (血清、血浆、组织匀浆液) | − | − | 5 |
| BCG试剂 | 250 | 250 | 250 |
4、白蛋白测定
先以空白孔调零，顺序加入BCG试剂，并立即混匀，室温放置 (30±3)s，在酶标仪628nm处读取标准孔和各测定孔的吸光度（即为A标准、A测定）。
5、(备选步骤)
当遇到脂血混浊，可设“样本空白孔”：取5μl待测样品加入250μl BCG空白试剂即为“样本空白孔”，以BCG空白试剂调零，读取“样本空白孔”的吸光度，用测定孔的吸光度减去“样本空白孔”的吸光度后的净吸光度，计算白蛋白浓度。
6、计算
白蛋白 (g/L)=A测定/A标准×白蛋白标准液浓度 (g/L)
参考区间
| 4～14岁儿童 | 38～54g/L |
| 健康成年人 | 34～48g/L |
注意事项
1、白蛋白标准粉末溶解于白蛋白标准配制液后，即获得白蛋白标准原液，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、如果没有酶标仪，也可以使用分光光度计测定，使用分光光度计测定蛋白浓度时，每个试剂盒可以测定的样品数量可能会显著减少。
3、本法线性范围是10～60g/L。
4、该法测定正常样本的批间变异系数为6.3%。
5、试剂开封后请尽快使用，以防影响后续实验效果。
Total Protein (TP) consists of albumin and globulin. Methods for albumin detection include the biuret method, tryptophan method, and dye-binding method. The dye-binding method for albumin can utilize Bromocresol Green (BCG) or Bromocresol Purple (BCP). These dyes have a high affinity for albumin. Typically, the initial rate of dye binding to albumin is monitored, which is proportional to the albumin concentration in the sample.
Assay Principle
Under acidic conditions, albumin molecules carry a positive charge and bind to the negatively charged Bromocresol Green (BCG), forming a blue-green complex. This complex has an absorption peak at 628 nm. The absorbance of this complex is proportional to the albumin concentration. By comparing it with a similarly processed albumin standard, the albumin concentration in the test sample can be determined. This method is commonly used for measuring protein content in human or animal serum, plasma, tissues, etc. It is simple, specific, and can be performed manually or using an automated analyzer. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
| A1505873 | Component | 100T | Storage |
| A1505873A | BCG Reagent | 25 mL | 2-8℃. Store in the dark. |
| A1505873B | Albumin Standard | 20 mg | RT |
| A1505873C | Albumin Standard Preparation Solution | 2 mL | RT |
| A1505873D | BCG Blank Reagent | 10 mL | RT |
Required Materials Not Provided
Centrifuge tubes, Small test tubes, Microplate reader, 96-well plate
Experimental Procedure
1. Preparation of Albumin Standard Solution
Add 0.5 mL of Albumin Standard Preparation Solution (or diluent) to the Albumin Standard (20 mg). Dissolve completely to prepare a 40 mg/mL Albumin Standard Stock Solution.
The solution can be used immediately after preparation.
Store the dissolved Albumin Standard Stock Solution at -20°C.
Important Note: The albumin standard should be dissolved in the same diluent used for the test samples. For example, if the test protein is dissolved in sucrose, dissolve the albumin standard in sucrose. Generally, 0.9% NaCl or PBS can also be used as the diluent for the albumin standard.
2. Sample Preparation
Serum/Plasma Samples: Use directly, take 5 μL for assay.
Tissue Samples: Homogenize the tissue in ice-cold physiological saline or PBS at a ratio of 1:9 (tissue weight (g) : saline volume (mL)). Centrifuge the homogenate at 2500 g for 10 minutes. Use 5 μL of the supernatant for assay.
3. Albumin Assay Setup
Set up Blank, Standard, and Test wells according to the table below.
Add reagents in the specified order, avoiding bubbles.
If the sample concentration is too high, reduce the sample volume or dilute appropriately before assaying. Duplicate wells are recommended.
| Reagent (μL) | Blank Well | Standard Well | Test Well |
| Albumin Standard Preparation Solution | 5 | − | − |
| Albumin Standard Solution (40 mg/mL) | − | 5 | − |
| Test Sample (Serum, Plasma, Tissue Homogenate) | − | − | 5 |
| BCG Reagent | 250 | 250 | 250 |
4. Albumin Measurement
Zero the microplate reader with the Blank well.
Add the BCG Reagent sequentially to all wells and mix immediately.
Incubate at room temperature for (30 ± 3) seconds.
Read the absorbance of the Standard well and Test wells at 628 nm (recorded as A<sub>standard</sub> and A<sub>test</sub>).
5. (Alternative Step) Handling Lipemic/Turbid Samples
For lipemic or turbid samples, set up a "Sample Blank" well:
Add 5 μL of test sample to 250 μL of BCG Blank Reagent. This is the "Sample Blank" well.
Zero the instrument with the BCG Blank Reagent.
Read the absorbance of the "Sample Blank" well (A<sub>sample blank</sub>).
Use the net absorbance (Atest - Asample blank) to calculate the albumin concentration.
6. Calculation
Albumin (g/L) = (Atest / Astandard) × Albumin Standard Concentration (g/L)
*(Where the Albumin Standard Concentration is 40 g/L if using the prepared stock solution directly in the calculation, but note the standard well contains a diluted amount. Ensure consistency in calculation based on the standard curve or direct ratio as implied. The formula provided assumes a direct proportionality between the absorbance in the test well and the standard well, using the known standard concentration.)*
*Clarification: If the standard well contains 5μL of 40 mg/mL standard, this is 0.2 mg of albumin in 255μL total volume. The calculation essentially compares the absorbance produced by the sample to that produced by a known amount of standard under identical conditions.*
| Children (4-14 years) | 38～54g/L |
| Healthy Adults | 34～48g/L |
Precautions
After dissolving the Albumin Standard powder in the Albumin Standard Preparation Solution, the resulting stock solution contains preservatives, which do not affect the assay. This stock solution can be stored long-term at -20°C.
If a microplate reader is unavailable, a spectrophotometer can be used. Note that the number of samples that can be assayed per kit may be significantly reduced when using a spectrophotometer.
The linear range of this method is 10 - 60 g/L.
The inter-assay coefficient of variation (CV) for normal samples using this method is 6.3%.
Use reagents promptly after opening to avoid affecting subsequent experimental results.
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838692 |
分析证书 | A1505873 |
| F2610353 |
分析证书 | A1505873 |
| F2608282 |
分析证书 | A1505873 |
| ZJ26F0636041 |
分析证书 | A1505873 |
| E2621774 |
分析证书 | A1505873 |
| ZJ26F0131757 |
分析证书 | A1505873 |
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