LabAssay LDL-Cholesterol Technical Information and Data

Kit Performance

Sample	Serum, Plasma
Target animals	Mouse, Rat
Calibration curve range	9.38-300 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-LDL cholesterol）

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

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

When Reacting Solution is added, the cholesterol and its derivatives in LDL produce hydrogen peroxide by CHE and CO. Hydrogen peroxide produced by the enzyme reactions with LDL-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 LDL-cholesterol contained in the sample is determined by measuring the absorbance of the blue color.

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	160	46.0	141	48.3	119	33.3
2	156	43.8	138	47.9	114	34.1
3	158	44.7	138	49.5	119	33.3
4	166	44.5	139	48.5	112	34.9
5	159	45.7	136	50.6	113	33.3
mean	160	44.9	138	49.0	115	33.8
SD	3.8	0.90	1.8	1.1	3.4	0.72
CV (%)	2.4	2.0	1.3	2.2	2.9	2.1

Result: The CV (%) of human serum was 2.0-2.4%, mouse serum was 1.3-2.2%, and rat serum was 2.1-2.9%, 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	125	88.1	41.8	119	91.4	48.4	130	78.7	45.2
2	124	87.0	41.1	119	88.1	45.0	129	76.1	42.9
3	130	91.5	41.7	118	88.0	48.2	126	74.2	49.7
4	125	89.5	39.8	126	95.1	50.4	131	74.5	49.4
mean	126	89.0	41.1	121	90.7	48.0	129.0	75.9	46.8
SD	2.7	1.9	0.92	3.7	3.4	2.2	2.2	2.1	3.3
CV (%)	2.1	2.2	2.2	3.1	3.7	4.7	1.7	2.7	7.1

Result: The CV (%) of human serum was 2.1-2.2%, mouse serum was 3.1-4.7%, and rat serum was 1.7-7.1%, 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	-	75.2	-	-
	7.12	82.3	7.10	100
	28.5	103	27.8	97.5
	35.6	112	36.8	103
Plasma (EDTA)	-	84.5	-	-
	10.6	93.8	9.30	87.7
	21.2	107	22.5	106
	26.5	112	27.5	104

Mouse

	Amount spiked (mg/dL)	Measured value (mg/dL)	Recovery volume (mg/dL)	Recovery rate (%)
Serum	-	86.6	-	-
	15.9	102	15.4	96.9
	23.8	112	25.4	107
	39.7	126	39.4	99.2
Plasma (EDTA)	-	97.7	-	-
	14.4	112	14.3	99.3
	28.9	126	28.3	97.9
	36.1	133	35.3	97.8

Rat

	Amount spiked (mg/dL)	Measured value (mg/dL)	Recovery volume (mg/dL)	Recovery rate (%)
Serum	-	14.6	-	-
	13.7	28.7	14.1	103
	41.0	53.0	38.4	93.7
	68.4	77.1	62.5	91.4
Plasma (EDTA)	-	16.2	-	-
	16.0	31.9	15.7	98.1
	48.1	64.4	48.2	100
	80.2	90.6	74.4	92.8

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	189	187	188	1.4	0.75
	2	88.8	93.2	91	3.1	3.4
	3	85.7	85.7	85.7	0	0
	4	99	93.9	96.5	3.6	3.7
	5	95.8	94	94.9	1.3	1.3
Plasma (EDTA)	1	122	121	122	0.71	0.58
	2	88.3	88	88.2	0.21	0.24
	3	85.9	84.4	85.2	1.1	1.2
	4	93.9	97.2	95.6	2.3	2.4
	5	95.1	95.4	95.3	0.21	0.22

Mouse

	No.	Measured value (mg/dL)		mean (mg/dL)	SD	CV (%)
Serum	1	32.6	33.4	33	0.57	1.7
	2	33.6	31.3	32.5	1.6	5
	3	33.4	32.4	32.9	0.71	2.1
	4	34.5	33.4	34	0.78	2.3
	5	36.1	34.1	35.1	1.4	4
Plasma (EDTA)	1	21.2	20.8	21	0.28	1.3
	2	16	17.7	16.9	1.2	7.1
	3	25.5	21.2	23.4	3	13
	4	19.1	20.3	19.7	0.85	4.3
	5	18.9	19.6	19.3	0.49	2.6

Rat

	No.	Measured value (mg/dL)		mean (mg/dL)	SD	CV (%)
Serum	1	43.3	37.9	40.6	3.8	9.4
	2	44.4	42.3	43.4	1.5	3.4
	3	42.7	44	43.4	0.92	2.1
	4	44.9	43.2	44.1	1.2	2.7
	5	50	51.1	50.6	0.78	1.5
Plasma (EDTA)	1	30.7	30.1	30.4	0.42	1.4
	2	33.7	32.8	33.3	0.64	1.9
	3	39.2	36.5	37.9	1.9	5
	4	30.7	30.9	30.8	0.14	0.46
	5	33.9	36.7	35.3	2	5.6