DESCRIPTION

Used for the quantitative determination of Anti-Nivolumab concentration in serum and plasma.

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Nivolumab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Nivolumab will be captured by immobilized Nivolumab. After washing away any unbound substances, a biotin-labeled Nivolumab is added to the wells. 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 proportion to the amount of Anti-Nivolumab 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): <10% Three samples of known concentration were tested sixteen times on one plate to assess intra-assay precision. Inter-Assay Precision (Precision between assays): <15% Three samples of known concentration were tested in twenty four separate assays to assess inter-assay precision.

Sample		1	2	3	1	2	3
n		16	16	16	24	24	24
Mean (ng/mL)		101.1	25.5	6.8	110.2	27.1	7.6
Standard deviation		3.8	0.8	0.6	4.0	1.3	0.5
CV \(%\)		3.7	3.3	8.1	3.6	4.6	6.7

RECOVERY

The recovery of Anti-Nivolumab 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=3)	1:10	92	81-111
	1:20	87	80-92

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Nivolumab and diluted with the Standard Diluent to produce samples with values within the dynamic range of the assay.

Sample	Dilution factor	Average (%)	Range (%)
Human serum (n=3)	1:2	105	104-107
	1:4	113	108-120
	1:8	110	107-112
	1:16	110	97-120