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。
胆固醇（Cholesterol）又称胆甾醇，是一种环戊烷多氢菲的衍生物，广泛存在于动物体内，其中脑、神经组织最丰富，在肾、脾、皮肤、肝和胆汁中含量也较高，用酶学方法测定总胆固醇（Total Cholesterol, TC）是生化检测中的常用方法，其特点是：1、灵敏度、准确度、精密度均高；2、使用温和的反应条件；3、操作简便；4、适用于自动分析仪。该试剂盒仅用于科研领域，不适用于临床诊断或其他用途。
检测原理：血液中的胆固醇约1/3为游离胆固醇，2/3为与脂肪酸结合的胆固醇酯，后者被胆固醇酯酶（CEH）水解为游离胆固醇，游离胆固醇被胆固醇氧化酶（COD）氧化成胆甾烯酮，并产生过氧化氢，再经过氧化物酶（POD）催化，使4-氨基安替比林与酚（三者合称PAP）反应，生成红色醌亚胺色素（Trinder反应），酶标仪在500~ 520nm处进行比色测定，用于人或动物的血清、血浆、脑脊液、细胞、组织等样本中的总胆固醇含量定量测定。
| T1505557 | Component | 100T | Storage |
| T1505557A | COD-PAP工作液 | 30 mL | -20℃. Store in the dark. |
| T1505557B | TC标准 (5mmol/L) | 1 mL | -20℃. Store in the dark. |
| T1505557C | ddH₂O | 1 mL | RT. |
自备仪器和试剂
操作步骤（仅供参考）
1、样本处理
1.1 血清、血浆、脑脊液样本
从待测样本中分离出的血清或血浆不应有溶血，直接检测，如超过线性范围，用生理盐水稀释后检测。
1.2 细胞样本
(1) 取适量的细胞（一般推荐>10⁶ 以上)，1000g 离心10min，弃上清，留取沉淀。
(2) 用PBS或生理盐水清洗1~2次，1000g 离心10min，弃上清，留取沉淀。
(3) 加入200~ 300μL的PBS或生理盐水匀浆，冰浴条件下超声破碎细胞，功率300W，每次3~5s，间隔30s，重复3~5次；亦可手动匀浆，制备好的匀浆液不可离心；亦可用1~2% Triton X-100冰浴30~ 60min，制备好的裂解液不可离心。
1.3 组织样本
准确称取适量组织样本，按质量 (g) : 生理盐水或PBS (ml) =1: 9的比例，加入生理盐水或PBS，冰浴条件下手动或机械匀浆，2500~3000g离心10min，取上清。
2、TC测定
2.1 各种仪器按下表依次加入试剂，充分混匀，37°C水浴中孵育5min。
酶标仪、全自动生化分析仪TC测定：
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分光光度计（1mL比色杯）、半自动生化分析仪TC测定：
|
普通分光光度计（2mL比色杯）TC测定：
|
2.2 立即用相应仪器测定500~ 520nm吸光度，以空白孔（管）调零，读取标准孔（管）、测定孔（管）的吸光度，分别记为A标准、A测定。
机器参数：
| 主波长/次波长 | 500/600nm |
| 反应类型 | 终点法 |
| 反应方向 | 升反应（+） |
3、计算公式
血清、血浆等液体样本（空白调零）：
TC (mmol/L)=A测定/A标准×5
血清、血浆等液体样本（全自动生化分析仪）：
TC (mmol/L)= (A测定-A空白)/(A标准-A空白)×5
组织样本（空白调零）：
TC (mmol/g)= A测定/A标准×5×V2/(m×1000)
组织样本（全自动生化分析仪）：
TC (mmol/g)= (A测定-A空白)/(A标准-A空白)×5×V2/(m×1000)
细胞样本（空白调零）：
TC (mmol/L)= A测定/A标准×5×V2/V1
细胞样本（全自动生化分析仪）：
TC (mmol/L)=(A测定-A空白)/(A标准-A空白)×5×V2/V1
参数说明
m：组织样本取样量，g
V1：细胞样本取样量，ml
V2：样本匀浆液总体积，ml
参考区间
健康成年人理想范围：<5.2 mmol/L (<200mg/dl)
边缘升高：<5.23~5.69 mmol/L (201~219mg/dl)
升高：25.72 mmol/L (≥220mg/dI)
备注: TC标准 (5mmol/L)=442.48 mg/dl
注意事项
1、上述低温试剂避免反复冻融，以免失效或效率下降。
2、COD-PAP工作液如不经常使用，应充分溶解后分装-20℃保存，可4°C短期保存。
3、本法可直接用于检测脑脊液中的TC含量，但不能直接检测尿液中的TC含量，因为未经处理的尿液中含有还原性物质，影响过氧化物酶反应。
4、检测TC的血清或血浆宜用EDTA或肝素抗凝，如不能及时测定，密闭保存，4℃可稳定1周，-20℃可以稳定半年以上。
5、本法线性范围可达13 mmol/L，如果样本TC浓度过高，结果可能呈假性降低，应用生理盐水稀释后重测，结果乘以稀释倍数。
6、该试剂盒既可作终点法检测，又可作速率法检测。
7、本法不适于检测游离胆固醇的浓度。
8、试剂开封后请尽快使用，以防影响后续实验效果。
Cholesterol, also known as cholesterin, is a derivative of cyclopentanoperhydrophenanthrene that is widely present in animals. It is most abundant in the brain and nervous tissue, and also found in relatively high levels in the kidneys, spleen, skin, liver, and bile. The enzymatic method for determining Total Cholesterol (TC) is commonly used in biochemical assays due to its characteristics: 1. High sensitivity, accuracy, and precision; 2. Use of mild reaction conditions; 3. Simple operation; 4. Suitable for automated analyzers. This kit is intended for research use only and is not suitable for clinical diagnosis or other purposes.
Detection Principle
In blood, approximately one-third of cholesterol exists as free cholesterol, while two-thirds is cholesterol ester bound to fatty acids. Cholesterol esters are hydrolyzed to free cholesterol by cholesterol esterase (CEH). Free cholesterol is then oxidized by cholesterol oxidase (COD) to cholestenone, producing hydrogen peroxide. Subsequently, in the presence of peroxidase (POD), 4-aminoantipyrine reacts with phenol (collectively referred to as PAP) to form a red quinoneimine dye (Trinder reaction). The absorbance is measured at 500–520 nm using a microplate reader for the quantitative determination of total cholesterol in samples such as human or animal serum, plasma, cerebrospinal fluid, cells, and tissues.
| T1505557 | Component | 100T | Storage |
| T1505557A | COD-PAP Working Solution | 30 mL | -20℃. Store in the dark. |
| T1505557B | TC Standard (5 mmol/L) | 1 mL | -20℃. Store in the dark. |
| T1505557C | ddH₂O | 1 mL | RT. |
Reagents, consumables and Equipments not provided
Operating Steps (For Reference Only)
1. Sample Preparation
1.1 Serum, Plasma, Cerebrospinal Fluid Samples
Freshly separated serum or plasma should be free of hemolysis. Test directly. If exceeding the linear range, dilute with physiological saline before testing.
1.2 Cell Samples
(1) Take an appropriate number of cells (generally recommended >10⁶), centrifuge at 1000 g for 10 minutes, discard the supernatant, and retain the pellet.
(2) Wash 1–2 times with PBS or physiological saline, centrifuge at 1000 g for 10 minutes, discard the supernatant, and retain the pellet.
(3) Add 200–300 μL of PBS or physiological saline for homogenization. Sonicate on ice (power 300 W, 3–5 s per pulse, 30 s intervals, repeat 3–5 times). Alternatively, homogenize manually. Do not centrifuge the prepared homogenate. Alternatively, lyse with 1–2% Triton X-100 on ice for 30–60 minutes. Do not centrifuge the prepared lysate.
1.3 Tissue Samples
Accurately weigh an appropriate amount of tissue. Add physiological saline or PBS at a mass (g) to volume (mL) ratio of 1:9. Homogenize manually or mechanically on ice. Centrifuge at 2500–3000 g for 10 minutes and collect the supernatant.
2. TC Measurement
2.1 For various instruments, add reagents in order according to the tables below. Mix thoroughly and incubate at 37°C for 5 minutes.
For Microplate Reader / Fully Automatic Biochemical Analyzer:
|
For Spectrophotometer (1 mL cuvette) / Semi-automatic Biochemical Analyzer:
|
For Standard Spectrophotometer (2 mL cuvette):
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朱传侠2.2 Immediately measure the absorbance at 500–520 nm using the corresponding instrument. Zero with the blank well/tube. Read the absorbance of the standard and test wells/tubes, recorded as A<sub>Standard</sub> and A<sub>Test</sub>, respectively.
Instrument Parameters:
|
3. Calculation Formulas
For liquid samples (serum, plasma, etc.) with blank zeroing:
TC (mmol/L) = ATest / AStandard × 5
For liquid samples (serum, plasma, etc.) with fully-automatic analyzer:
TC (mmol/L) = (ATest – ABlank) / (AStandard – ABlank) × 5
For tissue samples with blank zeroing:
TC (mmol/g) = ATest / AStandard × 5 × V2 / (m × 1000)
For tissue samples with fully-automatic analyzer:
TC (mmol/g) = (ATest – ABlank) / (AStandard – ABlank) × 5 × V2 / (m × 1000)
For cell samples with blank zeroing:
TC (mmol/L) = ATest / AStandard × 5 × V2 / V1
For cell samples with fully-automatic analyzer:
TC (mmol/L) = (ATest – ABlank) / (AStandard/– ABlank) × 5 × V2 / V1
Parameter Explanation
m: Mass of tissue sample taken (g)
V1: Volume of cell sample taken (mL)
V2: Total volume of sample homogenate (mL)
Reference Interval
Ideal range for healthy adults: <5.2 mmol/L (<200 mg/dL)
Borderline high: 5.23–5.69 mmol/L (201–219 mg/dL)
High: ≥5.72 mmol/L (≥220 mg/dL)
Note: TC Standard (5 mmol/L) = 442.48 mg/dL.
Precautions
1. Avoid repeated freeze-thaw cycles for the above low-temperature reagents to prevent loss of efficacy or reduced efficiency.
2. If the COD-PAP Working Solution is not used frequently, aliquot and store at –20°C after complete dissolution. It can be stored short-term at 4°C.
3. This method can directly measure TC in cerebrospinal fluid but not in urine, as untreated urine contains reducing substances that interfere with the peroxidase reaction.
4. Serum or plasma for TC measurement should be anticoagulated with EDTA or heparin. If not tested immediately, store sealed at 4°C (stable for 1 week) or at –20°C (stable for over 6 months).
5. The linear range of this method is up to 13 mmol/L. If the sample TC concentration is too high, results may be falsely low. Dilute the sample with physiological saline and re-assay, multiplying the result by the dilution factor.
6. This kit can be used for both endpoint and kinetic assays.
7. This method is not suitable for detecting free cholesterol concentration.
8. Use the reagents as soon as possible after opening to prevent affecting subsequent experimental results.
| T1505557 | 组件名称 | 100T | 储存条件 |
| T1505557A | COD-PAP工作液 | 30 mL | -20℃. Store in the dark. |
| T1505557B | TC标准 (5mmol/L) | 1 mL | -20℃. Store in the dark. |
| T1505557C | ddH₂O | 1 mL | RT. |
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