DESCRIPTION

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

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Pertuzumab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Pertuzumab will be captured by immobilized Pertuzumab. After washing away any unbound substances, a biotin-labeled Pertuzumab 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-Pertuzumab 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)		23.1	5.7	1.3	24.1	5.8	1.3
Standard deviation		0.8	0.3	0.1	2.7	0.5	0.2
CV \(%\)		3.5	4.9	8.9	11.2	8.8	13.7

RECOVERY

The recovery of Anti-Pertuzumab 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	85-119
	1:20	96	81-113

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Pertuzumab 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	99	87-108
	1:4	98	90-104
	1:8	99	88-110
	1:16	93	81-103