Competitive ELISA for quantification of trastuzumab.
Tissues from BT474 tumor mice that received trastuzumab injections were homogenized in 1 ml of 0.1 M Glycine pH2 with 1% Tween-20 and protease inhibitors followed by a centrifugation (4˚C, 10 min, 14,000 rpm). Six hundred microliters of supernatant was collected, and added with 150 ¦Ìl of 1 M Tris pH8 and 22.5 ¦Ìl of 5 M NaCl to neutralize the pH. Microtiter wells coated with 5 mg/ml rabbit anti-human IgG were incubated with the tissue extracts and 1 ¦Ìg/ml of biotinylated human IgG. After 2 hours of incubation at room temperature, the wells were washed with PBS containing 0.01% Tween 20, added with streptavidin-conjugated horseradish peroxidase, and incubated for 30 minutes at room temperature. The amount of biotinylated human IgG captured on the microtiter wells was quantified with 2,2-azino-bis(3-etylbenzthiazoline-6-sulfonic acid) as a substrate and the absorbance at 405 nm was measured. To obtain standard curves, the tissue extracts were substituted with various concentrations of trastuzumab (ranging from 0.01 to 10 ¦Ìg/ml) in the ELISA. |