DESCRIPTION

Neutrophil elastase (NE) is a major proteinase in primary granules in neutrophils that participates in microbicidal activity. NE (Leukocyte elastase, EC 3.4.21.37), a serine endopeptidase, is characterized by serine in the active tripeptide catalytic site: Asp, His, Ser. NE is the most abundant of four serine proteases present in neutrophils, which also include proteinase 3, cathepsin G, and neutrophil serine protease 4 (NSP4). NE is a 29.5 kD protein stored in mature form in the azurophilic granules of neutrophils and is present at high concentrations per azurophilic granule-approximately 67,000 molecules per granule.

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	1,249.0	47.7	3.8
2	8	264.5	8.8	3.3
3	8	139.5	4.1	2.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,228.0	60.3	4.9
2	16	242.7	24.5	10.1
3	16	128.5	12.8	10.0

RECOVERY

The recovery of mouse Neutrophil Elastase spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:32	95	87-109
Tissue lysate	1:4	103	83-117

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 mouse serum was initially diluted 1:2)

		Mouse serum(Sample Diluent PT 1-ef)	Tissue lysate(Sample Diluent PT 5-ef)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	106	111
	Range (%)	105-106	98-124
1:8	Average% of Expected	111	101
	Range (%)	108-114	89-113
1:16	Average% of Expected	100	88
	Range (%)	99-102	77-99

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse Neutrophil Elastase produced at Sunlong Medical™.