specifically interacts with the naturally epcam kato iii cell line with an affinity comparable to that of moc31 in contrast the antibody mt201 demonstrated significantly weaker binding to the epcam positive kato iii cells to determine the ability of 3 17i to bind to epcam western blot analysis elisa and biacore assays were performed to analyze the binding specificity and cross reactivity of antibodies 3 17i mt201 and moc31 to human and cynomolgus epcam sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page and western blot analysis were performed under reducing and non reducing conditions all the antibodies were in igg format and were produced in transiently transfected hek 293 cells the antibodies were then purified on protein a sepharose ge healthcare uppsala sweden followed by fractionation on two serial coupled size exclusion columns superdex 200 and superdex 75 the resultant igg preparations appeared to be monomeric with gt 95 purity the recombinant human and cynomolgus epcam antigens were also produced in hek 293 cells and purified in one step on a protein a sepharose column the determined purity of the antigen preparation was above 90 both epcam constructs consist of the extracellular domains of epcam fused to a human fc region from igg1 the human epcam sequence was derived from ncbi databse.