DESCRIPTION

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

PRINCIPLE OF THE ASSAY

This assay employs the quantitative sandwich enzyme immunoassay technique. Trastuzumab has been pre-coated onto a microplate. Samples or standards are pipetted into microwells and Anti-Trastuzumab will be captured by immobilized Trastuzumab. After washing away any unbound substances, a biotin-labeled Trastuzumab 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-Trastuzumab 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)		92.9	25.0	10.2	89.4	23.1	10.5
Standard deviation		4.2	1.4	0.8	8.8	1.9	0.9
CV \(%\)		4.5	5.6	7.9	9.9	8.3	8.2

RECOVERY

The recovery of Anti-Trastuzumab 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	100	94-111
	1:20	99	90-106

LINEARITY

To assess the linearity of the assay, human serum samples were spiked with high concentrations of Anti-Trastuzumab 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	98	94-101
	1:4	104	100-111
	1:8	101	94-113
	1:16	95	83-101