DESCRIPTION

CCL4(C-C chemokine ligand 4), also known as MIP-1β, plays a key role in inflammation and immune regulation. CCL4 is a major HIV-suppressive factor produced by CD8+ T cells. CCL4 is produced by a wide variety of cells including: immune cells (monocytes, B and T cells), fibroblasts, endothelial, and epithelial cells. CCL4 has a broad spectrum of target cells including immature dendritic cells, which express the cognate receptor CCR5. Increased expression of CCL4 was reported in the monocytes and blood of elderly individuals. Concentration of CCL4 within the body increases with age, which may cause chronic inflammation and liver damage.

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	502.9	19.4	3.9
2	20	121.2	3.5	2.9
3	20	61.9	1.6	2.6

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	486.5	26.2	5.4
2	24	117.0	3.2	2.7
3	24	60.8	2.9	4.8

RECOVERY

The recovery of mouse CCL4 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell culture supernatant	1:128	103	96-110
	1:256	99	85-109

LINEARITY

To assess the linearity of the assay, RAW 264.7 mouse monocyte/macrophage cell culture supernatant was diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell culture supernatant was diluted 1:32 prior to assay)

		Cell culture supernatant
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	102
	Range (%)	93-107
1:8	Average% of Expected	113
	Range (%)	93-125
1:16	Average% of Expected	113
	Range (%)	103-123

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CCL4 produced at Sunlong Medical™.