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。
检测原理：采用分光光度法以亚铁嗪为底物进行铁的检测，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，其他样品中的铁在酸性介质环境下也会被解离，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，适用于检测血清、血浆、组织等样品中的铁含量；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。
产品组分表
I1505783
| Component
| 50T
| Storage
|
I1505783A
| 铁标准 (100μg/mL) | 1 mL | 2-8℃. Store in the dark.
|
I1505783B
| 铁标准稀释液 | 10 mL
| RT
|
I1505783C
| Fe Assay Buffer | 60 mL
| 2-8℃
|
I1505783D
| 亚铁嗪显色液 | 3 mL
| 2-8℃. Store in the dark.
|
I1505783E
| ddH₂O | 10 mL
| RT |
自备仪器和试剂
离心管或试管、分光光度计、比色杯
实验流程
1、（选做）制备样品
1.1 血浆、血清样品
血浆、血清按照常规方法制备，-20℃冻存，用于Fe的检测。
1.2 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，-20℃冻存，用于Fe的检测。
1.3 高浓度样品
如果样品中含有较高浓度的Fe，可以使用 ddH₂O 稀释，不宜使用普通蒸馏水稀释。
注：（选做）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Fe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、制备铁标准工作液
取适量的铁标准 (100μg/mL)，按铁标准 (100μg/mL)：铁标准稀释液=1:49的比例配制铁标准 (2μg/mL)，即为铁标准工作液；4℃避光保存，3个月有效。
3、Fe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。
| 试剂（mL） | 空白管 | 标准管
| 测定管
|
ddH₂O
| 0.45 | —
| —
|
铁标准 (2μg/mL)
| —
| 0.45
| —
|
待测样品
| —
| —
| 0.45
|
Fe 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，
4、Fe检测
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、计算
5.1 血浆铁、血清铁
={[A测定−(A血清空白×0.97)] /A标准}×35.8 (单位：μmol/L)
={[A测定−(A血清空白×0.97)] /A标准}×2 (单位：mg/L)
5.2 组织铁、细胞铁
={[A测定−(A血清空白×0.97)] /A标准}×35.8/Pr (单位：μmol/L)
={[A测定−(A血清空白×0.97)] /A标准}×2 (单位：mg/L)
={[A测定−(A血清空白×0.97)] /A标准}×35.8/(Pr×N×V样÷V样总) (单位：μmol/N个)
={[A测定−(A血清空白×0.97)] /A标准}×2/(N×V样÷V样总) (单位：mg/N个)
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
Pr：待测样本蛋白浓度（g/L）
N：细胞或细菌总数
V样：反应体系中待测样品加入量，L
V样总：样品提取液总体积，L
单位换算：
铁标准 (2μg/mL)=铁标准 (2mg/L)=铁标准 (35.8μmol/L)
μg/dl=μmol/L/0.179
μmol/L=0.179*μg/dl=17.9*μg/ml
参考区间：
成年健康人血清铁：男性：11~30μmol/L (60~170μg/dl)，女性：9~27μmol/L (50~150μg/dl)
6、标准曲线参考
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：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。
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、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%。
9、试剂开封后请尽快使用，以防影响后续实验效果。
10、为了您的安全和健康，请穿实验服并戴一次性手套操作。
Iron is an essential trace element in the human body, with a total content of approximately 3270 mg. It is widely distributed: 67.6% of iron is found in hemoglobin as a prosthetic group, involved in oxygen transport; 2.59% and 4.15% are present in bone and myoglobin, respectively; and storage iron accounts for about 25.37%. Serum iron is bound to transferrin in the form of ferric ions (Fe³⁺). Therefore, measuring serum iron requires first dissociating Fe³⁺ from transferrin.
Assay Principle
This assay utilizes a spectrophotometric method with Ferrozine as the chromogen. In an acidic medium, serum iron bound to transferrin is released. Iron in other samples is also liberated under acidic conditions. The released iron is then reduced to ferrous ions (Fe²⁺) by a reducing agent (present in the assay buffer). The Fe²⁺ subsequently reacts with Ferrozine to form a magenta-colored complex. The absorbance at 562 nm is measured using a spectrophotometer. This method is suitable for detecting iron content in samples such as serum, plasma, and tissues. As this direct detection method is used, a serum blank should be included to correct for the sample's inherent color. The iron content is calculated according to the formula. This kit shows a good linear relationship up to 140 μmol/L. Triglycerides ≤ 3.39 mmol/L and bilirubin ≤ 171 μmol/L basically do not interfere with this method. This kit is for research use only and not suitable for clinical diagnosis or other purposes.
I1505783
| Component
| 50T
| Storage
|
I1505783A
| Iron Standard (100 μg/mL)
| 1 mL | 2-8℃. Store in the dark.
|
I1505783B
| Iron Standard Diluent
| 10 mL
| RT
|
I1505783C
| Fe Assay Buffer | 60 mL
| 2-8℃
|
I1505783D
| Ferrozine Chromogenic Solution
| 3 mL
| 2-8℃. Store in the dark.
|
I1505783E
| ddH₂O | 10 mL
| RT |
Required Materials Not Provided
Centrifuge tubes or test tubes, Spectrophotometer, Cuvettes
Experimental Procedure
1. (Optional) Sample Preparation
1.1 Plasma/Serum Samples
1.2 Cell or Tissue Samples
Homogenize an appropriate amount of cells or tissue.
Centrifuge at low speed and collect the supernatant.
Store at -20°C for Fe detection.
1.3 High-Concentration Samples
Note: (Optional) After preparation, determine the protein concentration using a BCA Protein Assay Kit to calculate Fe content per mg protein.
2. Preparation of Iron Standard Working Solution
3. Fe Assay Setup
Use dry test tubes or disposable sterile polyethylene centrifuge tubes cleaned with dilute hydrochloric acid and rinsed with deionized water.
Set up Blank, Standard, and Test tubes according to the table below.
Add reagents in the specified order, avoiding bubbles.
If the sample's iron concentration is too high, reduce the sample volume or dilute appropriately before assaying. Duplicate tubes are recommended.
Reagent (μL)
| Blank Tube
| Standard Tube
| Test Tube
|
ddH₂O
| 0.45 | —
| —
|
Iron Standard Working Solution (2 μg/mL)
| —
| 0.45
| —
|
Sample
| —
| —
| 0.45
|
Fe Assay Buffer
| 1.2 | 1.2
| 1.2 |
Mix well. Read absorbance at 562nm, zeroed with the Blank tube. Record for Test tube (This is the Serum Blank,
Ferrozine Chromogenic Solution | 0.05
| 0.05
| 0.05 |
4. Fe Measurement
Measure the absorbance of the Standard and Test tubes at 562 nm (recorded as Astandard and Atest).
Complete the colorimetric measurement within 1 hour.
5. Calculation
5.1 Plasma Iron, Serum Iron
Fe (μmol/L)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 35.8
Fe (mg/L)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 2
5.2 Tissue Iron, Cell Iron
Fe (μmol/g prot)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 35.8 / Pr
Fe (mg/g prot)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 2 / Pr
Fe (μmol/N cells)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 35.8 / (N × Vsample / Vtotal extract)
Fe (mg/N cells)
= { [Atest - (Aserum blank × 0.97)] / Astandard } × 2 / (N × Vsample / Vtotal extract)
Parameter Definitions:
Atest: Absorbance of the Test tube after adding Ferrozine Chromogenic Solution.
Aserum blank: Absorbance of the Test tube before adding Ferrozine Chromogenic Solution.
Astandard: Absorbance of the Standard tube.
Pr: Protein concentration of the test sample (g/L).
N: Total number of cells or bacteria.
Vsample: Volume of sample added to the reaction system (L).
Vtotal extract: Total volume of the sample extraction buffer (L).
Unit Conversions:
Reference Interval:
6. Standard Curve Reference
*(Placeholder for standard curve graph and data, typically showing absorbance vs. concentration for standards like 0, 0.5, 1, 2, 4, 6, 8, 10 μg/mL, measured at 562 nm)*
*Note: Standard curves may vary due to differences in instruments and operational techniques. Empirical data suggests deviations in the standard curve at iron standard concentrations below 0.1 μg/mL and above 40 μg/mL.*
Precautions
Hemolyzed samples interfere with the assay; avoid using them if possible.
If the sample concentration is too high, dilute with distilled water and re-assay, multiplying the result by the dilution factor.
Use high-purity deionized water during the experiment; do not use ordinary distilled water.
Glassware should be soaked in 10% hydrochloric acid for 24 hours and rinsed with deionized water before use.
Avoid contact with iron objects to prevent iron contamination.
The standard color is stable for 24 hours. The serum color is stable for 30 minutes; the color intensifies slowly over time, so measurement must be completed within 1 hour.
The value 0.97 is a volume correction factor.
The within-run coefficient of variation (CV) for this method is ≤ 3.1%; the between-run CV is ≤ 2.6%.
Use reagents promptly after opening to avoid affecting subsequent experimental results.
For your safety and health, wear lab coats and disposable gloves during operation.
J2615491
危险品化学品经营许可证（带存储）