DESCRIPTION

Reticulocalbin-2 (RCN2) is also named as endoplasmic reticulum Ca2+-binding protein of 55 kDa (ERC55) and E6-binding protein (E6BP). It is strictly located in endoplasmic reticulum. RCN2 is a low-affinity Ca2+ binding protein localized to the secretory pathway of mammalian cells, belongs to a member of the EF-hand Ca2+ binding protein family, which participate in the secretory process, chaperone activity, signal transduction as well as participation in a large variety of disease processes. As for cancer research, several studies showed that RCN2 was related to tumor progression, such as mammary cancer, cervical cancer, neck squamous cell carcinoma and colorectal cancer. RCN2 played a pivotal role in HCC pathogenesis through regulating activation of the epidermal growth factor receptor (EGFR) pathway and modulating epidermal growth factor receptor EGFR dimerization and internalization.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. 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 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	2,718.9	87.7	3.2
2	8	669.2	30.5	4.6
3	8	348.7	14.1	4.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,583.4	172.4	6.7
2	16	653.2	34.7	5.3
3	16	337.8	19.5	5.8

RECOVERY

The recovery of human RCN2 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:128	75	68-84
	1:256	80	76-86

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 cell lysate was initially diluted 1:32.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	105
	Range (%)	104-105
1:8	Average% of Expected	111
	Range (%)	105-117
1:16	Average% of Expected	111
	Range (%)	105-118

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human RCN2 Sandwich ELISA Kit produced at Sunlong Medical™.