DESCRIPTION

MCAM, also known as melanoma cell adhesion molecule (CD146) or MUC18, originally identified as a biomarker of melanoma progression, is a transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. Structurally, it consists of five Ig domains, a transmembrane domain, and a cytoplasmic region. A soluble form of CD146 has also been reported. In normal adult tissue, CD146 is primarily expressed by vascular endothelium and smooth muscle. CD146 is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis, and has been used as marker of circulating endothelium cells (CECs).

PRINCIPLE OF THE ASSAY

A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody also binds to the analyte. Horseradish peroxidase (HRP)-conjugated secondary antibody binds to the detection antibody. 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 (ng/mL)	SD	CV%
1	20	2,643.0	252.8	9.6
2	20	487.4	40.3	8.3
3	20	124.3	7.3	5.9

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	24	1,647.6	156.9	9.5
2	24	344.9	27.6	8.0
3	24	82.4	8.1	9.8

RECOVERY

The recovery of human CD146/MCAM spiked to three different levels throughout the range of the assay in vrious matrices was evaluated.

Sample Type		Average % of Expected	Range (%)
Human plasma	1:16	88	79-121
	1:32	95	82-118
Cell culture supernatant	1:4	96	82-125
	1:8	94	80-112

LINEARITY

To assess the linearity of the assay, three samples were spiked with high concentrations of human CD146/MCAM in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma	Cell culture supernatant
1:4	Average% of Expected	92	95
	Range (%)	83-92	83-109
1:8	Average% of Expected	93	106
	Range (%)	82-109	100-117
1:16	Average% of Expected	96	113
	Range (%)	82-114	110-115
1:32	Average% of Expected	94	100
	Range (%)	82-113	84-111

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CD146/MCAM Sandwich ELISA Kit produced at Sunlong Medical™.