Trastuzumab (Herceptin) Pharmacokinetic ELISA

The Trastuzumab ELISA kit is designed to measure free Trastuzumab with high specificity and sensitivity . This assay employs the sandwich enzyme immunoassay technique. A precoated anti-Trastuzumab 96 well plate is provided. Calibrator, quality control samples and test samples are pipetted into the appropriate wells. Trastuzumab present in biological matrices is bound by the immobilized capture antibody. After washing away any unbound substances, enzyme linked detection antibody is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of Trastuzumab present in test samples and the concentration is calculated from the standard series.

Coated microtiter plate, 96 wells Calibrator diluent. - 1.8ml Calibrator 12ul 10X wash buffer - 25ml Assay buffer - 50ml 1000X detection reagent - 17ul TMB - 12ml TMB stop solution - 12ml Plate sealers - 3

Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation: Dilute wash buffer concentrate with ultra-pure water 1/10 before use (for example add 10mL concentrate to 90mL ultra-pure water) . Mix well. 2. Detection Reagent (1X) Preparation: Dilute detection reagent with assay buffer 1/1000 before use (for example add 10μl concentrate to 10ml of assay buffer) . Mix welll. 3. Preparation of Calibrators: Prepare calibrators with concentrations ranging from 1000 ng/mL to 62.5 ng/mL. The following is an example calibrator curve.

This assay employs the sandwich enzyme immunoassay technique. Anti- Trastuzumab is coated onto a 96 well microplate. Calibrator, quality control samples (if desired) and test samples are pipetted into the appropriate wells. Trastuzumab present in biological matrices is bound by the immobilized anti- Trastuzumab antibody. After washing away any unbound substances, enzyme linked antiTrastuzumab antibody is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of Trastuzumab present in test samples. The color development is stopped and the intensity of the color is measured.

1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. Alternatively a log-log curve fit may be used. 2. The concentration of the unknowns can be read directly from this standard curve using the absorbance value for each sample. 3. Any sample undiluted or diluted still reading greater than the highest standard should be diluted appropriately with calibrator diluent and retested. If the samples have been diluted, the concentration determined from the standard curve must be multiplied by the dilution factor.

Dilute calibrators and test samples 1/50 with assay buffer (for example add 5µL of prepared calibrator or sample to 245µL of assay buffer) . Mix well. Do not store diluted samples.