DESCRIPTION

Chemokines direct the trafficking of white blood cells in immune surveillance, playing a key role in inflammatory and infectious diseases such as AIDS. CX3CL1, also known as fractalkine (FKN), is a unique, membrane-bound chemokine, with a transmembrane domain and the chemokine domain on top of a long mucinlike stalk. This protein can exist in two forms, a membrane-bound form or a shed soluble form, after extracellular proteolysis at a membrane-proximal dibasic cleavage site. The soluble CX3CL1 has potent chemoattractant activity for T cells and monocytes, and the cell-surface-bound protein, which is induced by primary proinflammatory signals in activated primary endothelial cells, promotes strong adhesion of those leukocytes. Presence of CX3CL1 and its receptor CX3CR1 in lymphocytes and their enhanced cellular expression during inflammatory conditions suggests that the CX3CL1-CX3CR1 duo participates in immune-related inflammatory diseases.

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 (ng/mL)	SD	CV%
1	8	4.48	0.17	3.79
2	8	1.13	0.06	5.31
3	8	0.62	0.02	3.23

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.71	0.28	5.94
2	16	1.20	0.10	8.33
3	16	0.64	0.04	6.25

RECOVERY

The recovery of mouse CX3CL1 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:4	99	93-104
	1:8	96	88-107
Tissue lysate	1:8	103	102-103
	1:16	113	108-120
Tissue homogenate	1:8	114	106-121
	1:16	108	104-113

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.

		Mouse serum(Sample Diluent PT 4B1)	Tissue lysate(Sample Diluent PT 3-ef)	Tissue homogenate(Sample Diluent PT 4B1)
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	108	93	88
	Range (%)	106-110	87-98	86-90
1:8	Average% of Expected	101	91	83
	Range (%)	94-108	80-103	74-92
1:16	Average% of Expected	80	87	78
	Range (%)	79-81	74-101	71-86

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CX3CL1 produced at Sunlong Medical™.