DESCRIPTION

CCL12, also known as MCP-5, is a small cytokine belonging to the CC chemokine family. It is a structural and functional homologue of human MCP-1. CCL12 is found predominantly in lymph nodes and thymus under normal conditions, and its expression can be markedly induced in activated macrophages both in vitro and in vivo. CCL12 is involved in allergic inflammation and the host response to pathogens and may play a pivotal role during early stages of allergic lung inflammation.

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	142.7	6.7	4.7
2	8	32.3	1.0	3.1
3	8	16.8	0.7	4.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	145.8	6.6	4.5
2	16	31.8	1.0	3.1
3	16	16.1	0.9	5.6

RECOVERY

The recovery of mouse CCL12/MCP-5 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:8	112	110-114
	1:16	100	95-106
Cell culture supernatant	1:8	108	103-110
	1:16	111	105-114

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 3)	Saliva(Sample Diluent PT 4B1)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	116	87
	Range (%)	106-127	82-92
1:8	Average% of Expected	100	83
	Range (%)	85-114	81-84
1:16	Average% of Expected	88	79
	Range (%)	72-104	77-80

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CCL12/MCP-5 One-Step ELISA Kit produced at Sunlong Medical™.