DESCRIPTION

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

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Tocilizumab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Tocilizumab will be captured by immobilized Tocilizumab. After washing away any unbound substances, a biotin-labeled Tocilizumab 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-Tocilizumab 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)		46.0	10.9	2.1	41.2	11.9	2.7
Standard deviation		1.6	0.3	0.2	3.8	1.4	0.3
CV \(%\)		3.5	3.2	9.3	9.3	12.0	10.6

RECOVERY

The recovery of Anti-Tocilizumab 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	98	82-110
	1:20	88	81-98

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Tocilizumab 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	102	99-106
	1:4	109	104-116
	1:8	111	107-116
	1:16	111	97-119