DESCRIPTION

E-cadherin is a transmembrane glycoprotein that connects epithelial cells at adherens junctions. In normal cells, E-cadherin exerts its tumor-suppressing role mainly by sequestering β-catenin from its binding to LEF (Lymphoid enhancer factor)/TCF (T cell factor), which serves the function of transcribing genes of the proliferative Wnt signaling pathway. The loss of E-cadherin expression associated with the epithelial-mesenchymal transition (EMT) occurs frequently during tumor metastasis. Furthermore, Soluble E-cadherin may be biomarkers for the diagnosis and prognosis of cancer.

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	648.2	23.4	3.6
2	8	158.1	2.7	1.7
3	8	82.8	1.7	2.1

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	656.6	22.8	3.5
2	16	160.6	4.8	3.0
3	16	81.5	2.3	2.8

RECOVERY

The recovery of mouse E-Cadherin spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:16,000	92	86-101
	1:32,000	96	86-107
Tissue lysate	1:640	87	85-89
	1:1,280	87	81-95

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. (The mouse serum was initially diluted 1:2,000. The tissue lysate was initially diluted 1:80.)

		Mouse serum(Sample Diluent PT 4B1)	Tissue lysate(Sample Diluent PT 4B1-ef)
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	103	107
	Range (%)	101-104	98-116
1:8	Average% of Expected	110	111
	Range (%)	107-114	108-113
1:16	Average% of Expected	109	123
	Range (%)	100-118	120-126

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse E-Cadherin produced at Sunlong Medical™.