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。
铁是人体必需微量元素，总含量约为3270mg，铁分布较广，有67.6%的铁作为血红蛋白分子的辅基分布于血红蛋白中，参与铁的运输；骨骼和肌红蛋白中各存在2.59%和4.15%，储存铁约占25.37%血清中铁均以三价铁离子形式与转铁蛋白结合，因此测定血清铁时，首先需要Fe³⁺与转铁蛋白分离；总铁结合力（TIBC）是指血清中转铁蛋白能与铁结合的总量。
检测原理：以亚铁嗪为底物进行总铁结合力的检测，将过量的铁加入待测血清、血浆中，使铁与多余的转铁蛋白结合，游离铁被铁吸附剂吸附除去，在酸性介质中与转铁蛋白结合的血清铁从转铁蛋白中解离出来，再被还原剂还原为Fe²⁺，后者与亚铁嗪生成紫红色化合物，通过分光光度计检测562nm处吸光度值，适用于检测血清、血浆样品中的总铁含量，即为总铁结合力（TIBC）；上述检测方法属于直接检测法，应设血清空白，纠正血清本身的色度，根据公式计算出铁含量，该检测试剂盒在140μmol/L以下线性关系良好，甘油三脂≤3.39mmol/L，胆红素≤171μmol/L，对本法基本无干扰。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
适用样本：血清、血浆
自备仪器和试剂
操作步骤
1、制备处理样品铁溶液和铁标准工作液
取适量的铁标准 (100μg/mL)，按铁标准 (100μg/mL)：TIBC铁标准稀释液=1：9的比例配制铁标准 (10μg/mL)，作为处理样品铁溶液； 同时按铁标准 (100μg/mL)：TIBC铁标准稀释液=1：49的比例配制铁标准 (2μg/mL)，作为铁标准工作液；4℃避光保存，3个月有效。
2、制备样品
血浆、血清按照常规方法制备，可以直接用于本试剂盒的测定，-20℃冻存，用于TIBC的检测，选用经稀盐酸处理及去离子水清洁的干燥有塞子的试管或者一次性无菌聚乙烯有盖子的离心管，加入血清或血浆0.45mL、铁标准 (10μg/mL) 0.25mL、ddH₂O 0.2mL，充分混匀，室温放置10min，加入铁吸附剂50mg，混匀，室温放置10min，期间振荡4次，3000g离心10min，取上清液，待用。
3、TIBC加样
选用经稀盐酸处理及去离子水清洁的干燥试管或者一次性无菌聚乙烯离心管，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；如果样品中的铁离子含量过高，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平行管。
加入物（mL） | 空白管 | 标准管 | 测定管 |
ddH₂O | 0.45 | — | — |
铁标准 (2μg/mL) | — | 0.45 | — |
上清液 | — | — | 0.45 |
TIBC Assay Buffer | 1.2 | 1.2 | 1.2 |
混匀，于562nm处，以空白管调零，读取测定管的吸光度（即血清空白，A血清空白）。
亚铁嗪显色液 | 0.05 | 0.05 | 0.05 |
4、TIBC测定
混匀，室温静置15min或37℃孵育10min，以空白管调零，分光光度计562nm处检测标准管、测定管的吸光度（即为A标准，A测定），1h内比色完毕。
5、结果计算
血浆、血清总铁结合力 (TIBC) (μmol/L)= {[A测定−(A血清空白×0.97)] /A标准}×71.6
参数说明：
A测定：测定管加入亚铁嗪显色液后测得的吸光度
A血清空白：测定管未加入亚铁嗪显色液前测得的吸光度
A标准：标准管的吸光度
单位换算
铁标准 (2μg/mL)=铁标准 (35.8μmol/L)
μg/dl=μmol/L/0.179
血浆、血清未饱和铁结合力 (UIBC) (μmol/L)=TIBC含量－血清铁含量
1、溶血样本对检测有干扰，尽量避免采用溶血样本。
2、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。
3、实验过程中用到的水，不可用普通的蒸馏水，尽量采用高纯度的去离子水。
4、玻璃器材需要10%的盐酸浸泡24h，取出后用去离子水冲洗后才可以使用。
5、避免与铁器接触，以防铁污染。
6、标准品呈色24h稳定，血清呈色30min内稳定，随着时间的延长，颜色会慢慢加深，应在1h内比色完毕。
7、0.97是体积校正值。
8、该法批内差异CV≤3.1%；批间差异CV≤2.6%。
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562nm对系列标准（0、0.5、1、2、4、6、8、10μg/mL）进行吸光度的测定，其标准曲线如下（仅供参考）：
注意：由于检测仪器和操作手法等条件的不同，标准曲线会有差异，该值仅供参考，根据测定经验显示铁标准在0.1μg/ml以下，40μg/ml以上，标准曲线会有偏差。
Iron is an essential trace element in the human body, with a total content of approximately 3270 mg. Iron is widely distributed; 67.6% of it exists as a cofactor in hemoglobin molecules, participating in oxygen transport. About 2.59% and 4.15% are found in bones and myoglobin, respectively, while stored iron accounts for approximately 25.37%. In serum, iron exists exclusively in the ferric (Fe³⁺) ion form bound to transferrin. Therefore, when measuring serum iron, it is first necessary to separate Fe³⁺ from transferrin. Total Iron-Binding Capacity (TIBC) refers to the total amount of iron that transferrin in the serum can bind.
Detection Principle
TIBC is detected using ferrozine as the substrate. An excess of iron is added to the test serum or plasma, allowing iron to bind to the available transferrin. Free iron is then removed by adsorption using an iron adsorbent. In an acidic medium, the serum iron bound to transferrin is released and reduced to Fe²⁺ by a reducing agent. The Fe²⁺ then reacts with ferrozine to form a purplish-red complex. The absorbance at 562 nm is measured using a spectrophotometer. This method is suitable for detecting the total iron content in serum and plasma samples, which represents the Total Iron-Binding Capacity (TIBC). This detection method is a direct assay and requires a serum blank to correct for the sample's inherent color. Iron content is calculated according to the formula. This detection kit shows a good linear relationship up to 140 μmol/L. Triglycerides ≤ 3.39 mmol/L and bilirubin ≤ 171 μmol/L cause minimal interference with this method. This kit is for research use only and is not suitable for clinical diagnosis or other purposes.
Applicable Samples: Serum, Plasma
Reagents, consumables and Equipments not provided
Operating Steps
1. Preparation of Sample Treatment Iron Solution and Iron Standard Working Solution
Take an appropriate amount of Iron Standard (100 μg/mL). Prepare the Iron Standard (10 μg/mL) by mixing Iron Standard (100 μg/mL) with TIBC Iron Standard Diluent at a ratio of 1:9. This serves as the Sample Treatment Iron Solution.
Simultaneously, prepare the Iron Standard (2 μg/mL) by mixing Iron Standard (100 μg/mL) with TIBC Iron Standard Diluent at a ratio of 1:49. This serves as the Iron Standard Working Solution.
Store at 4°C protected from light. Stable for 3 months.
2. Sample Preparation
Plasma and serum, prepared by conventional methods, can be directly used for detection with this kit. Store at -20°C for TIBC detection.
Use dry, stoppered test tubes or disposable sterile polyethylene centrifuge tubes with lids that have been treated with dilute hydrochloric acid and cleaned with deionized water.
Add 0.45 mL of serum or plasma, 0.25 mL of Iron Standard (10 μg/mL), and 0.2 mL of ddH₂O. Mix thoroughly and incubate at room temperature for 10 minutes.
Add 50 mg of Iron Adsorbent, mix, and incubate at room temperature for 10 minutes, vortexing 4 times during this period.
Centrifuge at 3000 g for 10 minutes. Collect the supernatant for use.
3. TIBC Sample Addition
Use dry test tubes or disposable sterile polyethylene centrifuge tubes treated with dilute hydrochloric acid and cleaned with deionized water.
Set up blank, standard, and test tubes according to the table below. Add solutions in the specified order, taking care to avoid bubbles. If the iron ion concentration in the sample is too high, reduce the sample volume or dilute appropriately before measurement. It is recommended to set up duplicate tubes for sample detection.
Substance Added (mL) | Blank Tube | Standard Tube | Test Tube |
ddH₂O | 0.45 | — | — |
Iron Standard (2 μg/mL) | — | 0.45 | — |
Supernatant | — | — | 0.45 |
TIBC Assay Buffer | 1.2 | 1.2 | 1.2 |
Mix well. Read the absorbance of the test tube at 562 nm using the blank tube for zeroing (this is the serum blank, ASerum Blank).
Ferrozine Chromogenic Solution | 0.05 | 0.05 | 0.05 |
4. TIBC Measurement
Mix well and let stand at room temperature for 15 minutes or incubate at 37°C for 10 minutes. Using the blank tube for zeroing, measure the absorbance of the standard and test tubes at 562 nm using a spectrophotometer (recorded as AStandard and ATest). Colorimetric measurement should be completed within 1 hour.
5. Result Calculation
Plasma/Serum Total Iron-Binding Capacity (TIBC) (μmol/L) = { [ATest − (ASerum Blank × 0.97)] / AStandard } × 71.6
Parameter Explanation:
ATest: Absorbance of the test tube measured after adding Ferrozine Chromogenic Solution.
ASerum Blank: Absorbance of the test tube measured before adding Ferrozine Chromogenic Solution.
AStandard: Absorbance of the standard tube.
Unit Conversion
Iron Standard (2 μg/mL) = Iron Standard (35.8 μmol/L)
μg/dL = μmol/L / 0.179
Plasma/Serum Unsaturated Iron-Binding Capacity (UIBC) (μmol/L) = TIBC − Serum Iron Content
Precautions
Hemolyzed samples interfere with detection; avoid using hemolyzed samples whenever possible.
If the sample concentration is too high, dilute with distilled water and repeat the measurement. Multiply the result by the dilution factor.
The water used in the experiment should not be ordinary distilled water; use high-purity deionized water whenever possible.
Glassware should be soaked in 10% hydrochloric acid for 24 hours, then rinsed with deionized water before use.
Avoid contact with ironware to prevent iron contamination.
The color of the standard is stable for 24 hours. The color of the serum sample is stable within 30 minutes. The color will gradually deepen over time, so measurement should be completed within 1 hour.
0.97 is the volume correction factor.
The intra-assay coefficient of variation (CV) for this method is ≤ 3.1%; the inter-assay CV is ≤ 2.6%.
Please use the reagent as soon as possible after opening to avoid affecting subsequent experimental results.
Appendix
Standard Curve Preparation:
Following the manual instructions at room temperature, measure the absorbance of a series of standards (0, 0.5, 1, 2, 4, 6, 8, 10 μg/mL) at 562 nm using a spectrophotometer. The standard curve is as follows (for reference only):
Note: Due to differences in detection instruments, operational techniques, and other conditions, the standard curve may vary. These values are for reference only. Based on testing experience, the standard curve may deviate for iron standards below 0.1 μg/mL and above 40 μg/mL.
| T1509330 | Component | 50T | Storage |
| T1509330A | 铁标准 (100μg/mL) | 3 mL | 2-8℃. Store in the dark. |
| T1509330B | TIBC铁标准稀释液 | 30 mL | RT. |
| T1509330C | TIBC Assay Buffer | 60 mL | 2-8℃. |
| T1509330D | 亚铁嗪显色液 | 3 mL | 2-8℃. Store in the dark. |
| T1509330E | ddH₂O | 10 mL | RT. |
| T1509330F | 铁吸附剂 | 3 g | RT. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0332423 |
分析证书 | T1509330 |
| ZJ26F0332424 |
分析证书 | T1509330 |
| ZJ26F0332425 |
分析证书 | T1509330 |
| ZJ26F0332426 |
分析证书 | T1509330 |
| ZJ26F0332427 |
分析证书 | T1509330 |
| ZJ26F0332428 |
分析证书 | T1509330 |
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