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  2. Life Science
  3. Metabolism Research
  4. ELISA / Assay Kits (Metabolism)
  5. Cholesterol Assay Kits
  6. LabAssay (TM) Free Cholesterol

LabAssay (TM) Free Cholesterol

for Cellbiology
Manufacturer :
FUJIFILM Wako Pure Chemical Corporation
Storage Condition :
Keep at 2-10 degrees C.
GHS :
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SDS
Comparison
Product Number
Package Size
Price
Inventory
Distributor
297-98501
Barcode No
4548995111142
350Tests
List Price
JPY 49,000

In stock in Japan

Please check here for notes on products and prices.

Document

SDS
Product Specification Sheet
Package Insert
Spectral Data
Certificate of Analysis
Calibration Certificate
Analytical Charts

Kit component

For 350 tests

Buffer Solution 75 mL/1 bottle
Chromogen Substrate For 75 mL/1 bottle
Standard Solution 2 mL/1 bottle

Product Overview

In human blood, cholesterol is found primarily in LDL and HDL, with smaller amounts present in VLDL. Approximately two-thirds of total cholesterol exists in esterified form, while the remaining one-third is present as free cholesterol. Free cholesterol refers to cholesterol that is not bound to a fatty acid and plays an essential role in maintaining cell membrane fluidity, signal transduction, and hormone synthesis. Maintaining an appropriate balance between free and esterified cholesterol is critical for normal cell function, and any disruptions in this balance may indicate the presence of various diseases.
The cholesterol esterification ratio typically ranges from 70–80%. A reduced ratio is associated with conditions such as acute and chronic hepatitis, liver cirrhosis, nephrotic syndrome, and chronic inflammatory conditions.

LabAssay™ Free Cholesterol is a kit designed to measure free cholesterol in samples. With the use of a microplate, this kit provides a convenient method for measuring free cholesterol in samples.

[Note] LabAssay™ series are reagents for research purposes. They cannot be used for diagnostic purposes.

Kit Performance

Analysis sample Human Serum/Plasma (EDTA, heparin)
Mouse Serum/Plasma (EDTA, heparin)
Rat Serum/Plasma (EDTA, heparin)
Dog Serum/Plasma (EDTA, heparin)
Cat Serum/Plasma (EDTA, heparin)
Culture medium (D-MEM, RPMI)*
Calibration curve range 10-233 mg/dL
Sample volume 10 μL
Measurement duration Approx. 20 min.
Wavelength 600 nm
* It is tested by using the samples that standard substances have been added to the culture medium.
Measurement availability depends on the culture medium, cell type and culture conditions. Please perform a spiked recovery test or dilution linearity test in advance using the culture medium to be used in your experiment.

Example of Calibration Curve

検量線例

Assay Principle

Upon addition of the color reagent to the sample, free cholesterol is oxidized by cholesterol oxidase (CO), generating hydrogen peroxide. The hydrogen peroxide then drives a quantitative oxidative condensation reaction between DAOS and 4-aminoantipyrine, catalyzed by peroxidase (POD), producing a blue pigment. The concentration of free cholesterol in the sample is determined by measuring the absorbance of the resulting blue color.

Data

Repeatability (within run precision)

Repeatability was conducted by measuring five-fold assays of serum/plasma (EDTA) samples from human at two concentrations with this product.

n＼ID Human serum Human plasma (EDTA)
ID1 (mg/dL) ID2 (mg/dL) ID3 (mg/dL) ID4 (mg/dL)
1 35.3 145 39.8 137
2 36.1 146 39.4 140
3 35.9 145 39.9 139
4 35.5 146 39.8 140
5 35.5 146 39.4 138
mean 35.7 146 39.7 139
SD 0.329 0.548 0.241 1.30
CV(%) 0.9 0.4 0.6 0.9

[Result]
The CV (%) of human serum was 0.4-0.9%, human plasma (EDTA) was 0.6-0.9%, indicating good repeatability.

Reproducibility (between run precision)

Reproducibility was conducted by measuring serum/plasma (EDTA) samples from human at two concentrations each for four days with this product.

Day＼ID Human serum Human plasma (EDTA)
ID9 (mg/dL) ID10 (mg/dL) ID11 (mg/dL) ID12 (mg/dL)
1 152 51.2 119 39.1
2 151 52.7 121 41.2
3 152 51.2 127 40.3
4 145 53.6 123 41.8
mean 150 52.2 123 40.6
SD 3.37 1.18 3.42 1.17
CV(%) 2.2 2.3 2.8 2.9

[Result]
The CV (%) of human serum was 2.2-2.3%, human plasma (EDTA) was 2.8-2.9%, indicating good reproducibility.

Dilution Linearity Test

Serum and plasma (EDTA) from human, mouse, rat, dog and cat, as well as D-MEM and RPMI spiked with the standard, were serially diluted twofold with the buffer in this kit and measured in duplicate to assess linearity.

Human serum
Human plasma （EDTA）
Mouse serum
Mouse plasma （EDTA）
Rat serum
Rat plasma （EDTA）
Dog serum
Dog plasma （EDTA）
Cat serum
Cat plasma （EDTA)
D-MEM
RPMI

[Result]
All measurement results showed good linearity within the measurement range.

Spike and Recovery Test

Standard solutions at three concentrations were added to serum/plasma (EDTA) from human, mouse, rat, dog and cat as well as culture medium (D-MEM and RPMI). Next, spike recovery tests were conducted, with each measurement performed in duplicate.

Human sample

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
Serum - 49.0 - -
13.8 62.5 13.5 97.8
27.7 77.5 28.5 103
55.3 101 52.0 94.0
Average 98.2
Plasma
(EDTA)		 - 41.3 - -
10.3 50.8 9.50 92.2
25.8 65.7 24.4 94.6
51.6 89.7 48.4 93.8
Average 93.5

Mouse sample (BALB/c)

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
Serum - 18.9 - -
12.6 30.6 11.7 92.9
25.2 44.1 25.2 100
50.3 72.5 53.6 107
Average 99.8
Plasma
(EDTA)		 - 17.8 - -
12.6 29.5 11.7 92.9
25.2 43.6 25.8 102
50.3 72.6 54.8 109
Average 101

Rat sample (S.D. rat)

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
Serum - 11.0 - -
11.1 21.2 10.1 91.0
22.1 31.4 20.4 92.3
55.3 64.3 53.3 96.4
Average 93.2
Plasma
(EDTA)		 - 10.4 - -
11.1 20.6 10.2 91.9
22.1 31.2 20.8 94.1
55.3 62.4 52.0 94.0
Average 93.3

Dog sample (Beagle)

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
Serum - 31.7 - -
11.4 42.4 10.7 93.9
20.6 50.3 18.6 90.3
51.4 82 50.3 97.9
Average 94.0
Plasma
(EDTA)		 - 29.6 - -
11.5 40.5 10.9 94.8
20.6 49.0 19.4 94.2
51.4 81.6 52.0 101
Average 96.7

Cat sample (Japanese cat)

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
Serum - 18.6 - -
11.4 29.2 10.6 93.0
20.6 37.5 18.9 91.7
51.4 70.2 51.6 100
Average 95.0
Plasma
(EDTA)		 - 18.8 - -
11.4 29.4 10.6 93.0
20.6 38.6 19.8 96.1
51.4 71.2 52.4 102
Average 97.0

Culture medium

Spiked amount
(mg/dL)		 Measurement value
(mg/dL)		 Recovery volume
(mg/dL)		 Recovery rate
(%)
D-MEM - 0.0 - -
10.6 10.8 10.8 102
21.2 21.1 20.1 94.8
53.0 52.0 52.0 98.1
Average 98.3
RPMI - 0.0 - -
10.6 11.1 11.1 105
21.2 20.5 20.5 96.7
53.0 51.8 51.8 97.7
Average 99.7

[Result]
Good recovery rates were confirmed.

Correlation with Automated Analyzer Reagents

Free cholesterol levels in serum/plasma (EDTA) samples from human were measured using LabAssay™ Free Cholesterol and reagents for automated analyzers, and the correlation between the two sets of results was evaluated.

Correlation with Automated Analyzer Reagents

[Result]
A strong correlation was confirmed between the values obtained with LabAssay™ Free Cholesterol and those obtained using reagents for automated analyzers.

Measurement Using Actual Samples

Mouse sample (BALB/c)

ID＼n Measurement value
(mg/dL)		 mean SD CV
(%)
1 2
Serum ID1 17.1 16.5 16.8 0.424 2.5
ID2 16.0 15.2 15.6 0.566 3.6
ID3 7.65 7.60 7.63 0.0354 0.5
ID4 21.8 21.2 21.5 0.424 2.0
ID5 33.8 33.4 33.6 0.283 0.8
Plasma
(EDTA)		 ID1 14.3 14.7 14.5 0.283 2.0
ID2 10.5 10.6 10.6 0.0707 0.7
ID3 15.4 15.5 15.5 0.0707 0.5
ID4 19.1 19.0 19.1 0.0707 0.4
ID5 30.8 30.7 30.8 0.0707 0.2

Rat sample (S.D. rat)

ID＼n Measurement value
(mg/dL)		 mean SD CV
(%)
1 2
Serum ID1 28.5 28.4 28.5 0.0707 0.2
ID2 17.6 17.5 17.6 0.0707 0.4
ID3 19.4 19.3 19.4 0.0707 0.4
ID4 28.4 28.2 28.3 0.141 0.5
ID5 19.0 19.0 19.0 0.00 0.0
Plasma
(EDTA)		 ID1 13.4 13.5 13.5 0.0707 0.5
ID2 12.4 12.2 12.3 0.141 1.1
ID3 14.5 13.9 14.2 0.424 3.0
ID4 28.5 28.3 28.4 0.141 0.5
ID5 19.5 19.2 19.4 0.212 1.1

Dog sample (Beagle)

ID＼n Measurement value
(mg/dL)		 mean SD CV
(%)
1 2
Serum ID1 28.2 27.3 27.8 0.636 2.3
ID2 19.5 19.2 19.4 0.212 1.1
ID3 27.8 25.1 26.5 1.91 7.2
ID4 28.8 28.5 28.7 0.212 0.7
ID5 18.4 18.6 18.5 0.141 0.8
Plasma
(EDTA)		 ID1 29.0 28.4 28.7 0.424 1.5
ID2 33.8 33.5 33.7 0.212 0.6
ID3 39.4 39.5 39.5 0.0707 0.2
ID4 26.3 25.8 26.1 0.354 1.4
ID5 17.0 17.0 17.0 0.00 0.0

Cat sample (Japanese cat)

ID＼n Measurement value
(mg/dL)		 mean SD CV
(%)
1 2
Serum ID1 22.6 21.9 22.3 0.495 2.2
ID2 14.5 14.4 14.5 0.0707 0.5
ID3 11.6 11.5 11.6 0.0707 0.6
ID4 15.3 15.4 15.4 0.0707 0.5
ID5 22.5 22.8 22.7 0.212 0.9
Plasma
(EDTA)		 ID1 21.9 21.4 21.7 0.354 1.6
ID2 10.2 10.3 10.3 0.0707 0.7
ID3 14.4 14.2 14.3 0.141 1.0
ID4 21.1 21.1 21.1 0.00 0.0
ID5 14.5 14.2 14.4 0.212 1.5

Example of Calculating the Cholesterol Esterification Ratio

The cholesterol esterification ratio in blood can be determined by using LabAssay™ Free Cholesterol (this product) in conjunction with LabAssay™ Cholesterol (Product No. 293-93601). The esterification ratio represents the percentage of cholesterol esters relative to total cholesterol, with cholesterol esters calculated as total cholesterol minus free cholesterol. The formula is shown below, along with representative calculations for human, mouse, rat, dog, and cat samples.

[Formula]

Human sample

Sample No. Total cholesterol*
(mg/dL)		 Free cholesterol
(mg/dL)		 Cholesterol esters
(mg/dL)		 Cholesterol esterification
ratio
Serum 1 119 36.0 83.0 69.7
2 163 48.2 115 70.5
3 152 47.3 105 68.9
Plasma
(EDTA)		 4 184 50.1 134 72.8
5 141 41.2 99.8 70.9
6 162 51.2 111 68.5

Mouse sample (BALB/c)

Sample No. Total cholesterol*
(mg/dL)		 Free cholesterol
(mg/dL)		 Cholesterol esters
(mg/dL)		 Cholesterol esterification
ratio
Serum 1 88.0 16.8 71.2 80.9
2 78.7 15.6 63.1 80.1
3 43.6 7.62 36.0 82.5
Plasma
(EDTA)		 4 33.7 14.5 19.2 57.0
5 45.0 10.6 34.4 76.5
6 35.9 15.5 20.4 57.0

Rat sample (S.D. rat)

Sample No. Total cholesterol*
(mg/dL)		 Free cholesterol
(mg/dL)		 Cholesterol esters
(mg/dL)		 Cholesterol esterification
ratio
Serum 1 93.6 28.4 65.2 69.6
2 64.6 17.5 47.1 72.9
3 65.7 19.3 58.1 70.5
Plasma
(EDTA)		 4 59.0 13.5 45.5 77.2
5 42.6 12.3 30.3 71.2
6 51.2 14.2 37.0 72.3

Dog sample (Beagle)

Sample No. Total cholesterol*
(mg/dL)		 Free cholesterol
(mg/dL)		 Cholesterol esters
(mg/dL)		 Cholesterol esterification
ratio
Serum 1 153 27.8 125 81.9
2 96.2 19.4 76.8 79.9
3 117 26.5 109 77.3
Plasma
(EDTA)		 4 129 28.7 100 77.8
5 142 33.6 108 76.3
6 173 39.5 134 77.2

Cat sample (Japanese cat)

Sample No. Total cholesterol*
(mg/dL)		 Free cholesterol
(mg/dL)		 Cholesterol esters
(mg/dL)		 Cholesterol esterification
ratio
Serum 1 103 22.2 80.8 78.5
2 81.5 14.4 67.1 82.3
3 91.5 11.6 83.9 87.4
Plasma
(EDTA)		 4 84.1 21.7 62.4 74.3
5 48.2 10.2 38.0 78.8
6 73.4 14.3 59.1 80.5
  • Total cholesterol was measured using our LabAssay™ Cholesterol (Product No. 293-93601).

FAQ

About sample

Which anticoagulants can I use?
Anticoagulants such as heparin, citrate and EDTA do not significantly influence the assay when used in normal amounts.
What should I do with samples that exceed the measurable range?
Dilute sample with saline and repeat the assay if the measured value exceeds the measurable range, and multiply the result by the dilution factor.
Is there any influence from substances that interfere with measurement (hemolysis, chyle, ascorbic acid, bilirubin, etc.)?
Please treat the measured values of hemolyzed samples as reference values. Chyle, ascorbic acid, and bilirubin have almost no effect on the measured values. We have confirmed that there is no influence at the following concentrations.

Effects of interfering substances

Hemolysis No influence up to 50 mg/dL
Chyle No influence up to 500 FTU
Ascorbic acid No influence up to 50 mg/dL
Bilirubin-F No influence up to 10 mg/dL
Bilirubin-C No influence up to 25 mg/dL

About kit usage

What instruments, and equipment are required for the assay using this kit?
The instruments and equipment required for the use of this kit are listed below.
  • 96-well microplate（transparent type）
  • Micropipette
  • Microtube
  • Pipette
  • Incubator maintained at 37°C
  • Plate mixer
  • Microplate reader with 600 nm wavelength filter
What is the amount of purified water to be added to the standard product?
Find and check "Reconstitution of standard" on this product page. As the amount of purified water to be added varies by lot, be sure to check it for every lot.
What are the storage conditions and shelf-life guidelines for each reagent?
Chromogen Reagent :
Prepare Chromogen Reagent by Dissolving 1 bottle of Chromogen Substrate （for 75 mL） to 75 mL of Buffer Solution. After reconstitution, the solution should be stored at 2°C - 10°C and used within 4 weeks.

Standard Solution :
Ready to use. After opening the bottle, store at 2°C - 10°C. * Standard solutions diluted to each concentration should be used immediately and must not be stored.

Overview / Applications

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For research use or further manufacturing use only. Not for use in diagnostic procedures.

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