LabAssay HDL-Cholesterol Technical Information and Data

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.

Data

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.

n＼ID	Human		Mouse		Rat
n＼ID	ID1 (mg/dL)	ID2 (mg/dL)	ID1 (mg/dL)	ID2 (mg/dL)	ID1 (mg/dL)	ID2 (mg/dL)
1	82.3	33.3	72.0	21.2	55.0	21.4
2	82.5	32.2	73.2	20.7	53.5	21.5
3	82.2	32.9	72.1	21.2	54.0	21.1
4	81.9	33.4	71.4	22.1	54.3	22.8
5	82.2	32.9	72.8	21.3	55.8	22.1
mean	82.2	32.9	72.3	21.3	54.5	21.8
SD	0.22	0.47	0.71	0.50	0.90	0.68
CV (%)	0.26	1.4	0.98	2.4	1.7	3.1

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.

Day＼ID	Human			Mouse			Rat
Day＼ID	ID1 (mg/dL)	ID2 (mg/dL)	ID3 (mg/dL)	ID1 (mg/dL)	ID2 (mg/dL)	ID3 (mg/dL)	ID1 (mg/dL)	ID2 (mg/dL)	ID3 (mg/dL)
1	79.2	47.0	25.2	77.7	44.6	26.3	56.0	39.7	19.0
2	77.6	45.4	23.6	74.2	42.2	25.2	58.4	39.6	18.2
3	80.5	48.8	24.8	79.9	45.2	26.8	60.5	41.5	19.0
4	79.2	46.9	25.1	80.5	44.4	26.5	59.1	40.5	18.8
mean	79.1	47.0	24.7	78.1	44.1	26.2	58.5	40.3	18.8
SD	1.2	1.4	0.74	2.9	1.3	0.70	1.9	0.88	0.38
CV(%)	1.5	3.0	3.0	3.7	3.0	2.7	3.2	2.2	2.0

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

	Amount spiked (mg/dL)	Measured value (mg/dL)	Recovery volume (mg/dL)	Recovery rate (%)
Serum	-	39.5	-	-
	11.3	50.3	10.8	95.6
	22.5	61.8	22.3	99.1
	28.2	67.1	27.6	97.9
Plasma (EDTA)	-	36.0	-	-
	7.72	43.8	7.80	101
	15.4	50.4	14.4	93.5
	19.3	54.3	18.3	94.8

Mouse

	Amount spiked (mg/dL)	Measured value (mg/dL)	Recovery volume (mg/dL)	Recovery rate (%)
Serum	-	11.7	-	-
	11.8	22.5	10.8	91.5
	35.4	47.0	35.3	99.7
	59.1	67.4	55.7	94.2
Plasma (EDTA)	-	10.5	-	-
	9.34	19.2	8.70	93.1
	28.0	36.6	26.1	93.2
	46.7	54.9	44.4	95.1

Rat

	Amount spiked (mg/dL)	Measured value (mg/dL)	Recovery volume (mg/dL)	Recovery rate (%)
Serum	-	15.6	-	-
	15.6	30.0	14.4	92.3
	31.2	45.0	29.4	94.2
	39.1	51.4	35.8	91.6
Plasma (EDTA)	-	13.0	-	-
	13.2	25.3	12.3	93.2
	19.9	32.8	19.8	99.5
	33.1	44.0	31.0	93.7

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

	No.	Measured value (mg/dL)		mean (mg/dL)	SD	CV (%)
Serum	1	58.2	57.9	58.1	0.2	0.37
	2	44.1	45.2	44.7	0.8	1.7
	3	45.7	45.7	45.7	0.0	0.0
	4	35.3	34.3	34.8	0.7	2.0
	5	34.9	34.3	34.6	0.4	1.2
Plasma (EDTA)	1	104	105	105	0.71	0.68
	2	44.7	45.5	45.1	0.57	1.3
	3	46.2	46.4	46.3	0.14	0.31
	4	34.5	35.3	34.9	0.57	1.6
	5	33.4	33.9	33.7	0.35	1.1

Mouse

	No.	Measured value (mg/dL)		mean (mg/dL)	SD	CV (%)
Serum	1	74.1	76.2	75.2	1.5	2.0
	2	71.9	71.7	71.8	0.14	0.20
	3	77.2	80.3	78.7	2.2	2.8
	4	86.5	87.7	87.1	0.85	0.97
	5	68.2	69.3	68.7	0.78	1.1
Plasma (EDTA)	1	40.6	40.3	40.5	0.21	0.52
	2	25.5	26.0	25.8	0.35	1.4
	3	50.1	48.6	49.4	1.1	2.1
	4	27.2	28.9	28.1	1.2	4.3
	5	24.2	25.7	25.0	1.1	4.3

Rat

	No.	Measured value (mg/dL)		mean (mg/dL)	SD	CV (%)
Serum	1	22.5	20.5	21.5	1.4	6.6
	2	31.8	30.7	31.3	0.78	2.5
	3	32.7	33.2	33.0	0.35	1.1
	4	22.4	22.5	22.5	0.071	0.31
	5	25.5	25.8	25.7	0.21	0.83
Plasma (EDTA)	1	13.2	13.3	13.3	0.071	0.53
	2	13.3	13.1	13.2	0.14	1.1
	3	13.1	11.4	12.3	1.2	9.8
	4	13.6	13.6	13.6	0.0	0.0
	5	15.4	16.4	15.9	0.71	4.4