DESCRIPTION

Cellular inhibitor of apoptosis protein 2 (cIAP2) is also named as baculoviral IAP repeat-containing protein 3 (BIRC3), hIAP-1, MIHC, RNF49 and Apoptosis inhibitor 2 (API2). cIAP2 is an essential member of IAPs family. Its combination with receptorinteracting protein kinase 1 (RIPK1) could activate NF-κb and MAPK pathways for cell survival. cIAP2 is a RING-containing protein ubiquitin ligase. cIAP2 normally inhibits antigen receptor signaling by mediating the ubiquitination and degradation of Bcl10, a critical component for antigenic signaling to NF-kappaB. cIAP2 as being highly expressed in SOX10-deficient drug tolerant persister (DTP) melanoma cells. cIAP2 is up-regulated in SOX10-deficient cells at the transcript level by the AP-1 protein complex component JUND. cIAP2 expressing cells can be targeted with the IAP inhibitor, birinapant, through induction of cell death. Many cancers express high levels of cIAP2 and other IAPs. For example, all four IAP family members were commonly elevated in prostate cancers, and cIAP2 expression was elevated in human colon cancer cells.

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	2,778.2	73.8	2.7
2	8	731.3	28.2	3.9
3	8	368.5	20.9	5.7

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,727.4	145.9	5.3
2	16	684.3	54.8	8.0
3	16	345.4	30.1	8.7

RECOVERY

The recovery of human cIAP2 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:64	118	117-120
	1:128	116	115-120

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 1:16.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	99
	Range (%)	92-104
1:8	Average% of Expected	95
	Range (%)	87-106
1:16	Average% of Expected	109
	Range (%)	100-121

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human cIAP2 Sandwich ELISA Kit produced at Sunlong Medical™.