DESCRIPTION

CCL11 also named Eotaxin, was initially discovered as an eosinophil-specific chemoattractant. CCL11 plays a central role in both allergic and non-allergic inflammatory reactions by recruiting immune cells such as eosinophils, basophils and Th2 lymphocytes. CCL11 binds to the chemokine receptors CCR2, CCR3 and CCR5, with highest affinity to CCR3. High levels of CCL11 have been described in several chronic inflammatory diseases, such as allergic rhinitis, atopic dermatitis, asthma, gastrointestinal disease and rheumatoid arthritis.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with biotin) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with biotin also binds to the analyte. Streptavidin-HRP binds to the biotin. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	20	226.2	14.3	6.3
2	20	57.5	2.7	4.6
3	20	13.8	0.6	4.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	242.1	21.0	8.7
2	24	57.5	2.8	5.0
3	24	15.4	0.5	3.1

RECOVERY

The recovery of mouse CCL11/Eotaxin spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:16	100	92-109
	1:32	96	79-108
Cell culture supernatant	1:2	112	103-122
	1:4	111	103-118

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentrations of mouse CCL11/Eotaxin in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Mouse serum were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The mouse serum samples were initially diluted 1:2)

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	100	106
	Range (%)	92-106	98-115
1:8	Average% of Expected	118	98
	Range (%)	116-120	93-104
1:16	Average% of Expected	122	105
	Range (%)	120-124	94-118

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CCL11/Eotaxin Sandwich ELISA Kit produced at Sunlong Medical™.