Generation of monoclonal antibodies to murine bile duct epithelial cells: Identification of annexin V as a new marker of small intrahepatic bile ducts

Biliary epithelial cells (BECs) are distributed along the length of both th e extrahepatic and intrahepatic biliary tree, but have distinctly different phenotypes and functions according to their anatomical location. It has be en reasoned that the distinct appearance of pathogenic lesions in different biliary diseases may be associated with the expression of distinct protein s. These data prompted us to immunize rats with cultured murine BECs with t he objective of determining if there are unique antigens on BECs. Of the 45 monoclonal antibodies (mAbs) produced, 12 mAbs (MBEC 1-12) were selected f or detailed study based on their classification into three major groups. Th ese groups included four antibodies (MBEC 1-4) that reacted in a staining p attern typical of mucin. A second group of mAbs, MBECs 5 to 8, reacted stro ngly along the biliary tract and by immunoblot analysis, reacted with sever al bands ranging from 44 kd to 64 kd, These antibodies were considered as m arkers of pan BECs and their staining pattern proved similar to that of a c ontrol polyclonal pan-cytokeratin. The final group of mAbs, MBECs 9 to 12, recognized a 36-kd antigen using lysates of murine BECs. These antibodies a lso predominantly stained small peripheral bile ducts. The reactive antigen was purified by immunoprecipitation and microsequenced; the peptides seque nced showed 100% homology with murine annexin V. The identification of anne xin V with predominantly intrahepatic bile ducts, is of significant interes t because of the multiple roles of annexin V, including that of membrane cy toskeletal interactions during transport and apoptosis.