DESCRIPTION

The chemokine CCL7 (MCP3) is a chemotactic factor and potent attractant of monocytes firstly characterized from the culture supernatants of MG-63 osteosarcoma cells. CCL7 is known to promote the recruitment of many innate immune cell types including monocytes and neutrophils to sites of bacterial and viral infection and eosinophils and basophils to sites of allergic inflammation. CCL7 is expressed at low levels in endothelial cells, fibroblasts and mononuclear cells and upregulated by various stimuli including viruses, type I or type II interferons (IFNs). CCL7 (MCP-3) mediates effects on a host of innate and adaptive immune cell types through binding to numerous receptors including CCR1, CCR2, CCR3, CCR5, and CCR10. Various studies have shown that tumor cells and stromal cells also produce high levels of CCL7, while the specific response element and signaling pathways involved are not entirely clear.

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	1,040.6	13.3	1.3
2	8	250.8	3.6	1.4
3	8	127.6	3.6	2.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,039.5	19.4	1.9
2	16	252.5	4.6	1.8
3	16	128.4	2.9	2.3

RECOVERY

The recovery of human CCL7/MCP-3 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	80	80-81
Cell culture supernatant	1:2	103	101-104
	1:4	101	98-106

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of human CCL7/MCP-3 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell culture supernatant samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human serum(Sample Diluent PT 4B1)	Cell culture supernatant(Sample Diluent PT 4B1-em)
1:2	Average% of Expected	89	100
	Range (%)	88-90	-
1:4	Average% of Expected	88	87
	Range (%)	84-91	85-89
1:8	Average% of Expected	104	72
	Range (%)	102-106	70-74
1:16	Average% of Expected	112	-
	Range (%)	106-118	-

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CCL7/MCP-3 One-Step ELISA Kit produced at Sunlong Medical™.