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。
| Component | 100T | Storage |
| Good's溶液 | 15 mL | 2-8℃. Store in the dark. |
| COD-PAP工作液 | 15 mL | -20℃. Store in the dark. |
| TC标准 (5mmol/L) | 1 mL | -20℃. Store in the dark. |
| ddH₂O | 1 mL | RT. |
自备仪器和试剂
1、生理盐水或PBS
2、96孔板或离心管、小试管、水浴锅或恒温箱、酶标仪或分光光度计、全自动或半自动生化分析仪
实验流程
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、试剂准备
临用前，按Good's溶液：COD-POD溶液=1：1混合，即为COD-POD工作液，4℃保存。
3、TC测定
3.1
酶标仪、全自动生化分析仪TC测定：
| 加入物（μL） | 空白孔 | 标准孔 | 待测孔 |
| ddH₂O | 3 | ||
| TC标准 (5mmol/L) | 3 | ||
| 待测样本 | 3 | ||
| COD-PAP工作液 | 300 | 300 | 300 |
分光光度计（1ml比色杯）、半自动生化分析仪TC测定：
| 加入物（μL） | 空白管 | 标准管 | 待测管 |
| ddH₂O | 0.01 | ||
| TC标准 (5mmol/L) | 0.01 | ||
| 待测样本 | 0.01 | ||
| COD-PAP工作液 | 1 | 1 | 1 |
普通分光光度计（2ml比色杯）TC测定：
| 加入物（μL） | 空白管 | 标准管 | 待测管 |
| ddH₂O | 0.02 | ||
| TC标准 (5mmol/L) | 0.02 | ||
| 待测样本 | 0.02 | ||
| COD-PAP工作液 | 2 | 2 | 2 |
各种仪器按上表依次加入试剂，充分混匀，37°C水浴中孵育5min。
3.2 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（＋） |
4、计算公式
血清、血浆等液体样本（空白调零）：FC (mmol/L)=A测定/A标准x5
血清、血浆等液体样本（全自动生化分析仪）：FC (mmol/L)= (A测定-A空白)/(A标准-A空白)x5
组织样本（空白调零）：FC (mmol/g)= A测定/A标准x5xV2/(mx1000)
组织样本（全自动生化分析仪）：FC (mmol/g)= (A测定-A空白)/(A标准-A空白)x5xV/(mx1000)
细胞样本（空白调零）：FC (mmol/L)= A测定/A标准x5xV2/V1
细胞样本（全自动生化分析仪）：FC (mmol/L)=(A测定-A空白)/(A标准-A空白)x5xV2/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: Approximately one-third of the cholesterol in blood is free cholesterol, and two-thirds is cholesterol esterified with fatty acids. The latter is hydrolyzed to free cholesterol by cholesterol esterase (CEH). The free cholesterol is then oxidized by cholesterol oxidase (COD) to cholesterone, producing hydrogen peroxide. Subsequently, under the catalysis of peroxidase (POD), 4-aminoantipyrine reacts with phenol (collectively known as PAP) to produce a red quinoneimine dye (Trinder reaction). The color intensity is measured colorimetrically at 500-520 nm using a microplate reader. This kit is used for the quantitative determination of total cholesterol content in serum, plasma, cerebrospinal fluid, cells, tissues, and other samples from humans or animals.
| Component | 100T | Storage |
| Good's Solution | 15 mL | 2-8℃. Store in the dark. |
| COD-PAP Working Solution | 15 mL | -20℃. Store in the dark. |
| TC Standard (5 mmol/L) | 1 mL | -20℃. Store in the dark. |
| ddH₂O | 1 mL | RT. |
User-Prepared Instruments and Reagents
Physiological saline or PBS
96-well plate or centrifuge tubes, small test tubes, water bath or incubator, microplate reader or spectrophotometer, fully automatic or semi-automatic biochemical analyzer
Experimental Procedure
1. Sample Preparation
1.1 Serum, Plasma, Cerebrospinal Fluid Samples
Serum or plasma separated from the test sample should not be hemolyzed. Assay directly. If the concentration exceeds the linear range, dilute with physiological saline before assaying.
1.2 Cell Samples
(1) Take an appropriate amount of cells (generally recommended >10⁶), centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.
(2) Wash the pellet 1-2 times with PBS or physiological saline, centrifuge at 1000 g for 10 min, discard the supernatant, keep the pellet.
(3) Add 200-300 μL of PBS or physiological saline to homogenize. Sonicate the cells on ice (power 300W, pulse 3-5s, interval 30s, 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 min. Do not centrifuge the prepared lysate.
1.3 Tissue Samples
Accurately weigh an appropriate amount of tissue sample. 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 min. Collect the supernatant for assay.
2. Reagent Preparation
Before use, mix the Good's solution and COD-POD solution at a ratio of 1:1 to prepare the COD-POD working solution, which should be stored at 4°C.
3. TC Assay
3.1 Assay Procedure
For Microplate Reader / Fully Automatic Biochemical Analyzer:
| Reagent (μL) | Blank Well | Standard Well | Test Well |
| ddH₂O | 3 | ||
| TC Standard (5 mmol/L) | 3 | ||
| Test Sample | 3 | ||
| COD-PAP Working Solution | 300 | 300 | 300 |
For Spectrophotometer (1 mL cuvette) / Semi-automatic Biochemical Analyzer:
| Reagent (μL) | Blank Tube | Standard Tube | Test Tube |
| ddH₂O | 0.01 | ||
| TC Standard (5 mmol/L) | 0.01 | ||
| Test Sample | 0.01 | ||
| COD-PAP Working Solution | 1 | 1 | 1 |
For Conventional Spectrophotometer (2 mL cuvette):
| Reagent (μL) | Blank Tube | Standard Tube | Test Tube |
| ddH₂O | 0.02 | ||
| TC Standard (5 mmol/L) | 0.02 | ||
| Test Sample | 0.02 | ||
| COD-PAP Working Solution | 2 | 2 | 2 |
Add reagents to the respective wells/tubes according to the tables above. Mix thoroughly and incubate in a 37°C water bath for 5 minutes.
3.2 Measurement
Immediately measure the absorbance at 500-520 nm using the corresponding instrument. Zero the instrument with the blank well/tube. Read the absorbance of the standard and test wells/tubes, recorded as Astandard and Atest, respectively.
Instrument Parameters Suggestion:
Primary Wavelength/Secondary Wavelength: 500/600 nm
Reaction Type: Endpoint
Reaction Direction: Increase (+)
4. Calculation Formulas
For Serum, Plasma and other liquid samples (Blank zeroed):
TC (mmol/L) = (Atest / Astandard) × 5
For Serum, Plasma and other liquid samples (Fully automatic biochemical analyzer):
TC (mmol/L) = [(Atest - Ablank) / (Astandard - Ablank)] × 5
For Tissue samples (Blank zeroed):
TC (mmol/g) = (Atest / Astandard) × 5 × V₂ / (m × 1000)
For Tissue samples (Fully automatic biochemical analyzer):
TC (mmol/g) = [(Atest - Ablank) / (Astandard - Ablank)] × 5 × V₂ / (m × 1000)
For Cell samples (Blank zeroed):
TC (mmol/10⁶ cells) = (Atest / Astandard) × 5 × V₂ / V₁
For Cell samples (Fully automatic biochemical analyzer):
TC (mmol/10⁶ cells) = [(Atest - Ablank) / (Astandard - Ablank)] × 5 × V₂ / V₁
Parameter Definitions:
m: Tissue sample weight (g)
V₁: Volume of cell sample taken (mL)
V₂: Total volume of sample homogenate (mL)
Reference Interval (Healthy Adults)
Desirable range: < 5.2 mmol/L (< 200 mg/dL)
Borderline high: 5.23 – 6.18 mmol/L (201 – 238 mg/dL)
High: ≥ 6.19 mmol/L (≥ 239 mg/dL)
Note: TC Standard (5 mmol/L) = 193.3 mg/dL (Note: 442.48 mg/dL in the original text appears to be a miscalculation; 5 mmol/L * 386.65 g/mol ≈ 1933 mg/L = 193.3 mg/dL)
Precautions
1. Avoid repeated freeze-thaw cycles for the low-temperature reagents mentioned above to prevent inactivation or decreased efficiency.
2. If the COD-PAP Working Solution is not used frequently, it should be completely dissolved, aliquoted, and stored at -20°C. It can be stored short-term at 4°C.
3. This method can be directly used to detect TC content in cerebrospinal fluid but cannot directly detect TC in urine, as untreated urine contains reducing substances that interfere with the peroxidase reaction.
4. Serum or plasma for TC detection should preferably be anticoagulated with EDTA or heparin. If not assayed 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 rate method detection.
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.
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