DESCRIPTION

Monocyte chemotactic protein 1 (MCP-1; also known as CCL2), is chemotactic for monocyte/macrophage, B-cell, and Tlymphocyte, and belongs to the CC subfamily of chemokines. Chemokines are a superfamily of secreted proteins involved in immunoregulatory and inflammatory processes. The human ortholog has been implicated in the pathogenesis of diseases characterized by monocytic infiltrates, such as psoriasis, rheumatoid arthritis, and atherosclerosis.

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	225.8	8.5	3.8
2	8	49.5	0.5	1.0
3	8	25.3	0.3	1.2

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	232.8	9.4	4.0
2	16	49.9	1.4	2.8
3	16	25.7	0.8	3.1

RECOVERY

The recovery of mouse MCP-1/CCL2 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:2	94	93-96
Cell culture supernatant	1:400	97	87-110
Tissue lysate	1:4	92	87-96

LINEARITY

To assess the linearity of the assay, mouse serum samples were spiked with high concentrations of mouse MCP-1/CCL2 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell culture supernatant and tissue lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell culture supernatant was initially diluted 1:50.)

		Mouse serum	Cell culture supernatant	Tissue lysate
1:2	Average% of Expected	98	100	100
	Range (%)	92-105	-	-
1:4	Average% of Expected	115	84	105
	Range (%)	111-120	77-91	100-111
1:8	Average% of Expected	106	90	107
	Range (%)	104-109	79-102	106-108
1:16	Average% of Expected	97	83	110
	Range (%)	90-105	76-90	89-130

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse MCP-1/CCL2 One-Step ELISA Kit produced at Sunlong Medical™.