DESCRIPTION

GPNMB also known as HGFIN, osteoactivin, and DC-HIL, is a type I membrane glycoprotein involved in various biological processes, including inflammation, invasion and metastasis of malignant tumors, cell differentiation, and tissue regeneration. GPNMB shows expression in the lowly metastatic human melanoma cell lines and xenografts but does not show expression in the highly metastatic cell lines. GPNMB acts as an osteogenic factor that stimulates osteoblast differentiation in vivo and in vitro.

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 (ng/mL)	SD	CV%
1	8	17.99	1.26	7.00
2	8	1.80	0.04	2.22
3	8	0.97	0.02	2.06

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	18.31	1.52	8.30
2	16	1.82	0.04	2.20
3	16	0.99	0.03	3.03

RECOVERY

The recovery of mouse GPNMB spiked to three different levels throughout the range of the assay in mouse serum was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:2	75	73-78

LINEARITY

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

		Mouse serum
1:2	Average% of Expected	101
	Range (%)	98-103
1:4	Average% of Expected	99
	Range (%)	97-101
1:8	Average% of Expected	103
	Range (%)	101-106
1:16	Average% of Expected	101
	Range (%)	99-103

CALIBRATION

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