Recombinant human epcam

Both antibodies were tested for their ability to recognize EpCAM on the surface of the human positive control cell lines, MCF-7 and MDA-MB-231. Antibodies (1:50 dilution of a 0.2 mg/ml stock or 4 μg/ml final concentration) or negative control rabbit serum or goat γ-globulin (at an equivalent concentration to the primary antibody) were incubated with cells for approximately 30 min and then washed three times with phosphate-buffered saline (PBS) and 1% bovine serum albumin (BSA). Cells were subsequently incubated with the appropriate FITC-conjugated secondary antibody (1:200 dilution, Invitrogen, a division of ThermoScientific), incubated for an additional 30 min, and washed three times in PBS-BSA. After washing, cells were resuspended in 300–500 μl of PBS and data was collected on a FACS Calibur (BD Biosciences; Franklin Lakes, NJ). Data was analyzed using Flowjo software (BD Biosciences; Ashland, OR). Once EpCAM expression on the human positive control breast cancer cell lines was confirmed for each antibody, the antibodies were then evaluated for binding to the feline cell lines using flow cytometry. For competition experiments, 5 × 10⁵ cells were incubated with 0.5 and 1.5 μg of recombinant human EpCAM (R&D Systems), then labeled with the primary antibody or negative control, as above.

Fmoc‐amino acids and coupling reagents were purchased from either Anaspec (Fremont, CA) or Novabiochem (Germany). PEG500 (NH₂‐(PEG)‐COOH, 0.5 kDa), PEG2000 (2 kDa), and PEG5000 (5 kDa) were purchased from Nektar Therapeutics (Huntsville, AL). G7 PAMAM dendrimers were purchased from Dendritech Inc. (Midland, MI). CellTracker Green and PBS solution were obtained from Thermo Fisher Scientific (Waltham, MA). Fetal bovine serum (FBS) and antibiotic mixture (penicillin/streptomycin) were acquired from Invitrogen. Dulbecco's modification of Eagle's medium (DMEM), DMEM/Ham's F‐12 50/50 mix (DMEM/F‐12), and Roswell Park Memorial Institute (RPMI) medium were purchased from Corning (Manassas, VA). F‐12 nutrient mixture (Ham's F12) and trypsin‐EDTA were obtained from Gibco (Grand Island, NY). Recombinant human EpCAM was purchased from R&D Systems. All other chemicals were obtained from Sigma‐Aldrich (St. Louis, MO), unless otherwise stated.