DESCRIPTION

Neutrophil gelatinase-associated lipocalin (NGAL) also known as Lipocalin-2 (LCN2), is a secreted glycoprotein that belongs to the lipocalin protein family. Several roles of NGAL was identified, including iron trafficking, chemotactic, and promote cell differentiation and proliferation. NGAL was originally found in human neutrophils, and expressed in tissues containing renal, uterus, liver, spleen, as well as cardiomyocytes, neurons and immune cells. NGAL is a significant biomarker of cardiovascular diseases, ischemic stroke, acute renal injury, inflammation and metabolic diseases, and it can be detected in human serum, saliva and urine.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the solution yellow. The color intensity is proportional to the quantity of bound protein, which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	598.1	13.1	2.2
2	8	221.8	8.9	4.0
3	8	96.6	1.6	1.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	599.1	13.1	2.2
2	16	220.8	6.7	3.0
3	16	95.3	2.4	2.5

RECOVERY

The recovery of human NGAL spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:400	91	88-95
	1:800	85	81-92
Cell culture supernatant	1:32	103	97-109
	1:64	93	84-99
Urine	1:200	98	97-99
	1:400	101	95-107
Saliva	1:1,600	100	97-104
	1:3,200	95	90-98

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human plasma was initially diluted 1:50. The cell culture supernatant was initially diluted 1:16. The urine was initially diluted 1:25. The saliva was initially diluted 1:200.)

		Human plasma	Cell culturesupernatant	Urine	Saliva
1:2	Average% of Expected	100	100	100	100
	Range (%)	-	-	-	-
1:4	Average% of Expected	78	100	94	94
	Range (%)	76-80	100-101	85-102	93-96
1:8	Average% of Expected	80	104	106	115
	Range (%)	79-81	102-106	95-118	108-122
1:16	Average% of Expected	102	110	103	83
	Range (%)	90-113	105-115	99-107	73-92

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human NGAL produced at Sunlong Medical™.