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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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