DESCRIPTION

Epithelial cell adhesion molecule (EPCAM), also known as CD326, is a well-characterized type I transmembrane glycoprotein that plays a pivotal role in cell adhesion, proliferation, differentiation, and signaling. It mediates Ca2+-independent homotypic cellcell adhesion through its extracellular domain, which is crucial for the formation and maintenance of epithelial tissues. The membrane localization of EPCAM is essential for its function in cell adhesion and signaling. Additionally, elevated levels of soluble EPCAM have been associated with tumor burden and poor prognosis in cancer patients, making it a potential biomarker for cancer diagnosis and monitoring.

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	511.8	8.9	1.7
2	8	132.4	2.6	2.0
3	8	36.7	1.0	2.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	521.2	13.2	2.5
2	16	130.8	3.6	2.8
3	16	36.6	0.9	2.5

RECOVERY

The recovery of human EPCAM/CD326 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:8	101	93-107
	1:16	106	103-108
Cell culture supernatant	1:32	106	93-122
	1:64	105	99-115
Urine	1:2	79	77-81
Cell lysate	1:3,200	112	104-123
	1:6,400	108	99-114

LINEARITY

To assess the linearity of the assay, urine samples were spiked with high concenthumanions of human EPCAM/CD326 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human serum, cell culture supernatant and cell lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The human serum was initially diluted 1:2. The cell culture supernatant was initially diluted 1:4. The cell lysate was initially diluted 1:400.)

		Human serum	Cell culturesupernatant	Urine	Cell lysate
1:2	Average% of Expected	100	100	88	100
	Range (%)	-	-	85-91	-
1:4	Average% of Expected	118	98	96	92
	Range (%)	114-122	97-99	91-100	91-93
1:8	Average% of Expected	122	95	100	91
	Range (%)	117-127	94-96	97-102	89-93
1:16	Average% of Expected	127	88	102	87
	Range (%)	120-134	80-95	97-107	84-90

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human EPCAM/CD326 One-Step ELISA Kit produced at Sunlong Medical™.