Total Cholesterol (TC) Content Assay Kit (Single-Reagent COD-PAP, Micro Method) - BioReagent, high purity

Cat. No.: T1505557

GRADE & PURITY BioReagent

Descripción general

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

• Microplate reader or spectrophotometer (capable of measuring absorbance at 500–520 nm)
• Fully-automatic or semi-automatic biochemical analyzer, water bath or incubator
• 96-well plate, centrifuge tubes, or small test tubes
• Physiological saline or PBS  

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:

Substance Added (μL)	Blank Well	Standard Well	Test Well
ddH₂O	3	—	—
TC Standard (5 mmol/L)	—	3	—
Sample	—	—	3
COD-PAP Working Solution	300	300	300

For Spectrophotometer (1 mL cuvette) / Semi-automatic Biochemical Analyzer:

Substance Added (mL)	Blank Tube	Standard Tube	Test Tube
ddH₂O	0.01	—	—
TC Standard (5 mmol/L)	—	0.01	—
Sample	—	—	0.01
COD-PAP Working Solution	1	1	1

For Standard Spectrophotometer (2 mL cuvette):

Substance Added (mL)	Blank Tube	Standard Tube	Test Tube
ddH₂O	0.02	—	—
TC Standard (5 mmol/L)	—	0.02	—
Sample	—	—	0.02
COD-PAP Working Solution	2	2	2

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_Standard and A_Test, respectively.

Instrument Parameters:

Parameter	Setting
Primary/Secondary Wavelength	500/600 nm
Reaction Type	Endpoint assay
Reaction Direction	Increasing (+)

3. Calculation Formulas

For liquid samples (serum, plasma, etc.) with blank zeroing:

TC (mmol/L) = A_Test / A_Standard × 5

For liquid samples (serum, plasma, etc.) with fully-automatic analyzer:

TC (mmol/L) = (A_Test – A_Blank) / (A_Standard – A_Blank) × 5

For tissue samples with blank zeroing:

TC (mmol/g) = A_Test / A_Standard × 5 × V₂ / (m × 1000)

For tissue samples with fully-automatic analyzer:

TC (mmol/g) = (A_Test – A_Blank) / (A_Standard – A_Blank) × 5 × V₂ / (m × 1000)

For cell samples with blank zeroing:

TC (mmol/L) = A_Test / A_Standard × 5 × V₂ / V₁

For cell samples with fully-automatic analyzer:

TC (mmol/L) = (A_Test – A_Blank) / (A_Standard/– A_Blank) × 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.

Almacenamiento y envío

Condiciones de almacenamiento de almacenamiento

Protected from light,Room temperature,Store at -20°C

Enviado en

Ice chest + Ice pads

Estabilidad y almacenamiento

Each component has a shelf life of 6 months under corresponding storage conditions.

Contents & Storage

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.