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International Immunology, Vol. 4, No. 7, pp. 789-796,July 1992
© 1992 Japanese Society for Immunology

Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies

Ludy Dobrila, Felicia Antohe, Constantina Heltianu and Maya Simionescu

Institute of Cellular Biology and Pathology 8, B. P. Hasdeu Street, Bucharest 79691. Romania

Correspondence to: Correspondence to: L. Dobrila

Studies were conducted to identify and localize albumin binding proteins (ABPs) in endothelial cells using rabbits polyclonal anti-idiotypic antibodies (Ab2) raised against the affinity-purified anti-bovine serum albumin IgG. Ab2 were purified by fast performance liquid chromatography. The anti-idiotypic nature of the IgG was assessed by (i) the capacity to inhibit albumin binding to its specific antibody in a dose-dependent manner, (ii) the lack of interaction with albumin, and (iii) the interaction with anti-albumin antibodies of diverse origins. The latter characteristic indicates that although polyclonal, the purified anti-idiotypic antibodies contain some of the Ab2ß type. The binding of Ab2 to cultured bovine aortic endothelial cell surfaces was saturable and specific as demonstrated by radloimmunoassay (RIA) and immunofluorescence studies respectively. A competitive RIA was used to test whether Ab2 competed for albumin binding to bovine aortic endothelial cells (BAECs) (presumably to ABPs). It was found that Ab2 inhibited binding of [125l]albumin to BAECs in a dose-dependent fashion. Immunobiot analysis of extracts of BAECs, microvascular endothelial cells, and lung showed that both Ab2 and albumin bind specifically to two polypeptldes with an apparent molecular mass of 18 and 31 kDa. In addition, upon radioiodination of BAECs apical membrane proteins, Ab2 bound specifically and immunoprecipltated restrictively two radiolabeled cell surface proteins of 18 and 31 kDa. The results provide direct evidence for the presence of the 18 and 31 kDa peptldes (ABPs) on the endothelial cell membrane and/or associated structures, i.e. open plasmalemmal vesicles and uncoated pits.

Keywords: cell culture, receptors, immunoprecipitation, ligand blotting, immunofluorescence

Received 8 April 1991, accepted 8 April 1992.


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