DESCRIPTION

Interleukin 8 (IL-8), also known as CXCL8, which is a member of the CXC chemokine family. This chemokine is secreted by a variety of cell types including monocyte/macrophages, T cells, neutrophils, fibroblasts, endothelial cells, and various tumor cell lines in response to inflammatory stimuli. IL-8 has two primary functions. It induces chemotaxis in target cells, primarily neutrophils but also other granulocytes, causing them to migrate toward the site of infection. IL-8 also induces phagocytosis once they have arrived. This gene is believed to play a role in the pathogenesis of bronchiolitis, a common respiratory tract disease caused by viral infection. IL-8 is also known to be a potent promoter of angiogenesis. IL-8 has been associated with tumor angiogenesis, metastasis, and poor prognosis in breast cancer. IL-8 may present a novel therapeutic target for estrogen driven breast carcinogenesis and tumor progression.

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	51.0	0.4	0.8
2	8	13.6	0.1	0.7
3	8	7.3	0.2	2.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	51.5	0.6	1.2
2	16	13.6	0.2	1.5
3	16	7.2	0.2	2.8

RECOVERY

The recovery of human/cynomolgus monkey CXCL8/IL-8 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	81	79-83
	1:4	90	89-92
Cynomolgus monkey serum	1:160	97	94-99
	1:320	98	97-100
Human cell culture supernatant	1:4,000	95	91-97
	1:8,000	100	99-102
Cynomolgus monkey cell culture supernatant	1:128	101	98-104

LINEARITY

To assess the linearity of the assay, human plasma samples were spiked with high concentrations of human/cynomolgus monkey CXCL8/IL-8 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cynomolgus monkey serum and cell culture supernatants samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cynomolgus monkey serum was initially diluted 1:20. The human cell culture supernatant was initially diluted 1:500. The cynomolgus monkey cell culture supernatant was initially diluted 1:256.)

		Human plasma	Cynomolgusmonkey serum	Human cell culturesupernatant	Cynomolgusmonkey cell culturesupernatant
1:2	Average% of Expected	77	100	100	100
	Range (%)	73-81	-	-	-
1:4	Average% of Expected	92	96	98	113
	Range (%)	91-93	94-99	97-98	111-115
1:8	Average% of Expected	96	93	99	107
	Range (%)	95-96	92-95	97-101	104-109
1:16	Average% of Expected	96	93	97	109
	Range (%)	92-100	90-97	95-99	107-111

CALIBRATION

The NIBSC/WHO Human CXCL8/IL-8 Reference Reagent (89/520) (rDNA derived), which was intended as a potency standard, was evaluated in this kit. The dose response curve of this Reference Reagent parallels the standard curve. To convert sample values obtained with the Human CXCL8/IL-8 ELISA kit to approximate NIBSC/WHO (89/520) values, use the equation below. NIBSC (89/520) approximate value (IU/mL)=0.005× Human CXCL8/IL-8 value (pg/mL)