LabAssay™ HDL-Cholesterol

Cholesterol circulates in the body in particles called lipoproteins. There are different types of lipoprotein, among which HDL-cholesterol is responsible for transporting excess cholesterol to the liver, from tissues, eliminated cells, and blood, preventing increased blood cholesterol levels, and suppressing abnormal lipid metabolism and atherosclerosis. A decrease in HDL-cholesterol concentration is seen in coronary artery disease, hyperlipidemia and hypocholesterolemia, smoking, obesity, diabetes, and liver disease. HDL is an important protective factor against coronary heart disease (CHD), with hypo-HDL cholesterolemia one of the major risk factors for CHD.

LabAssay™ HDL-Cholesterol is a kit for the determination of HDL-cholesterol in blood (serum and plasma). With the use of a microplate, this kit provides a quick and convenient method for measuring HDL-cholesterol in samples. In addition, our kit do not require fractionation of lipoproteins by centrifugation.

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

Kit Performance

Sample	Serum, Plasma
Target animals	Mouse, Rat
Calibration curve range	6.25-200 mg/dL
Sample volume	5 μL
Measurement duration	Approx. 20 min
Wavelength	Primary wavelength 600 nm Reference wavelength 700 nm

Example of Calibration Curve

Principle

1) The first reaction (elimination of non-HDL cholesterol)

The block polymer containing hydrophilic and hydrophobic moiety in Pretreatment selectively binds to HDL in the specimens, and protects it from enzyme (CHE, CO) reactions. CHE and CO react with non-HDL lipoprotein [chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL)]. Hydrogen peroxide produced by the enzyme reactions with non-HDL cholesterol is decomposed to water by catalase in Pretreatment reagent.

2) The second reaction (color reaction of HDL-cholesterol)

When Reacting Solution is added, the cholesterol and its derivatives in HDL produce hydrogen peroxide by CHE and CO. Hydrogen peroxide produced by the enzyme reactions with HDL-cholesterol yields a blue color pigment upon oxidative condensation with N-(3-sulfopropyl)-3-methoxy-5-methylaniline (HMMPS) and 4-aminoantipyrine in the presence of peroxidase (POD). The amount of HDL-cholesterol contained in the sample is determined by measuring the absorbance of the blue color.

Precision of assay (within assay variation)

Reproducibility was confirmed by performing quintuplicate assays of serum samples of two concentrations each from humans, mice, and rats with this product.

[Result]
The CV (%) of human serum was 0.26-1.4%, mouse serum was 0.98-2.4%, and rat serum was 1.7-3.1%, indicating good reproducibility.

Reproducibility (between assay variation)

Triplicate measurements were performed on human, mouse, and rat serum samples at three concentrations each for four days with this product.

[Result]
The CV (%) of human serum was 1.5-3.0%, mouse serum was 2.7-3.7%, and rat serum was 2.0-3.2%, indicating good reproducibility.

Dilution linearity test

Using the standard diluent supplied with the product, human serum/plasma (EDTA), mouse serum/plasma (EDTA), and rat serum/plasma (EDTA) at two different concentrations each, were subjected to a 2-fold serial dilution with sample buffer and measured to confirm linearity (measured in duplicates).

[Result]
Good linearity was confirmed for all samples within the calibration curve range.

Spiked recovery test

Standard solutions at three concentrations were added to human serum/EDTA plasma, mouse serum/EDTA plasma and rat serum/EDTA plasma. Next, spike-recovery tests were conducted, with each measurement performed in duplicate.

Human

Mouse

Rat

[Result]
Good recovery rates was confirmed for all samples.

Measurement examples

Human serum/plasma, mouse (ICR) serum/plasma samples, and rat (SD) serum/plasma were measured with this product.

Human

Mouse

Rat