DESCRIPTION

Used for the quantitative determination of Denosumab concentration in serum and plasma.

PRINCIPLE OF THE ASSAY

This assay employs the quantitative competitive enzyme immunoassay technique. Recombinant Human CD254 has been pre-coated onto a microplate. Standards or samples are premixed with biotin-labeled antibody and then pipetted into the wells. Denosumab in the sample competitively binds to the pre-coated protein with biotin-labeled Denosumab. After washing away any unbound substances, Streptavidin-HRP is added to the wells. Following a wash to remove any unbound enzyme reagent, a substrate solution is added to the wells and color develops in inversely proportion to the amount of Denosumab bound in the initial step. The color development is stopped and the intensity of the color is measured.

PRECISION

Intra-Assay Precision (Precision within an assay): <20% Three samples of known concentration were tested sixteen times on one plate to assess intra-assay precision. Inter-Assay Precision (Precision between assays): <20% Three samples of known concentration were tested in twenty four separate assays to assess inter-assay precision. Intra-Assay Precision Inter-Assay Precision Sample 1 2 3 1 2 3 n 16 16 16 24 24 24 Mean (ng/mL) 858.2 164.6 31.9 817.5 149.0 36.4 Standard deviation 103.2 29.9 4.9 76.8 22.8 5.2 CV (%) 12.0 18.2 15.3 9.4 15.3 14.3

RECOVERY

The recovery of Denosumab spiked to three different levels in human serum samples throughout the range of the assay was evaluated. Sample Dilution factor Average (%) Range (%) Human serum (n=4) 1:10 97 81-114 1:20 105 92-120

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Denosumab and diluted with the Standard Diluent to produce samples with values within the dynamic range of the assay. (Human serum samples were prediluted 10 fold) Sample Dilution factor Average (%) Range (%) Human serum (n=3) 1:2 87 83-91 1:4 90 80-100 1:8 89 83-100 1:16 89 81-100