DESCRIPTION

CCN4, also known as WISP-1, is a member of the WNT1 inducible signaling pathway protein (WISP) subfamily of the connective tissue growth factor/CCN family of matricellular proteins. CCN4 is both an intracellular and a secreted protein found in the extracellular matrix and is present throughout the body. Like many other matricellular proteins, CCN4 can also regulate cellular responses such as cell growth, differentiation and survival. Additionally, CCN4 plays a multiple physiologic roles in development and also participates in pathogenesis. CCN4 is associated with cancer and can serve as a biomarker or prognostic factor as well as a potential therapeutic target.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with biotin) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with biotin also binds to the analyte. Streptavidin-HRP binds to the biotin. 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 (pg/mL)	SD	CV%
1	20	459.3	15.1	3.3
2	20	116.0	3.3	2.8
3	20	25.6	1.4	5.4

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	24	466.8	20.0	4.3
2	24	117.2	4.3	3.6
3	24	25.5	1.7	6.7

RECOVERY

The recovery of human WISP-1 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:2	82	73-92
	1:4	91	77-99

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of human WISP-1 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human serum
1:2	Average% of Expected	91
	Range (%)	82-96
1:4	Average% of Expected	96
	Range (%)	88-102
1:8	Average% of Expected	93
	Range (%)	92-94
1:16	Average% of Expected	90
	Range (%)	87-93

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human WISP-1/CCN4 Sandwich ELISA Kit produced at Sunlong Medical™.