DESCRIPTION

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

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Ranibizumab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Ranibizumab will be captured by immobilized Ranibizumab. After washing away any unbound substances, a biotin-labeled Ranibizumab 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-Ranibizumab 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)		1980.3	509.3	119.3	2293.3	539.8	155.5
Standard deviation		110.1	28.4	6.3	201.6	60.0	20.7
CV \(%\)		5.6	5.6	5.3	8.8	11.1	13.3

RECOVERY

The recovery of Anti-Ranibizumab 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	85	81-90
	1:20	88	81-95

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Ranibizumab 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	99	96-104
	1:4	101	100-102
	1:8	106	102-111
	1:16	106	97-113