DESCRIPTION

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

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Rituximab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Rituximab will be captured by immobilized Rituximab. After washing away any unbound substances, a biotin-labeled Rituximab 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-Rituximab 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)		32.1	8.6	2.1	32.2	8.8	2.4
Standard deviation		0.8	0.2	0.1	1.7	0.3	0.2
CV \(%\)		2.5	2.3	4.2	5.1	3.0	8.1

RECOVERY

The recovery of Anti-Rituximab 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	91	80-112
	1:20	89	85-95

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Rituximab 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=4)	1:2	96	91-99
	1:4	107	104-111
	1:8	118	114-120
	1:16	106	100-113