Pergamon 0300-9629(95)02002-2 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA C am p . Bio c he m . Phy sio l. Vol. llZA, Nos. 314, pp. 347-354. 1995 Copyright 0 1995 Elsevier Science Inc. Printed in Great Britain. All rights reserved 0300-%29/95 $9.50 + .oa Monoclonal antibodies to chicken ovotransferrin: epitopic and phylogenetic analysis Anne B. Mason,* Chantal J. Kenney,* Michael K.Miller,* Robert C. Woodworth,* Kokila J. Pate17 and Robert W. Evans? *Department of Biochemistry, University of Vermont, College of Medicine, Burlington, VT 05405 USA; and tDepartment of Biochemistry and Molecular Biology, United Medical and Dental Schools of Guy’s and St. Thomas’s Hospitals, Guy’s Hospital, London SE1 9RT, England zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 1. The ability of four domain-specific anti-chicken ovotransferrin antibodies to bind to turkey, quail, duck, pheasant and goose ovotransferrins was examined directly by an enzyme-linked immunoassay and indirectly in a competitive radioimmunoassay. 2. The ability of these same ovotransferrins to compete with radioiodinated chicken ovotransferrin for binding to transferrin receptors on chick embryo red blood cells was also tested. 3. The hypothesis that evolutionarily conserved determinants involved in receptor recognition can be predicted by monoclonal antibodies that block binding of ovotransferrin to receptor was found to be incorrect. Key words: Ovotransferrin; Monoclonal antibody; Epitope; ELISA; Solid-phase radioimmunoassay; Transfer-tin receptor; Binding studies. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Comp. Biochem. Physiol. 112A, 347-354, 1995. Introduction Iron is required for cell division. In cells grow- ing in tissue culture, much of the iron is ob- tained from the 80 kDa serum protein trans- ferrin, which holds two ferric ions. The iron-laden transferrin binds to specific recep- tors on the cell surface; the receptor- transferrin complex is internalized by recep- tor-mediated endocytosis, the iron dissociates from the transferrin and the iron free protein, still bound to receptor, is returned to the cell surface. There, at neutral pH, it diffuses into the external milieu, freeing the receptor to bind more diferric transferrin (Klausner ef al., 1983). The exact mechanism by which iron is removed remains elusive, although it appears to involve changes in pH and large conforma- tional changes in both the transferrin and the C o rre sp o nde nc e to : Dr. Anne B. Mason, Department of Biochemistry, University of Vermont, College of Medicine, Burlington VT 05405 USA. Received 8 February 1995; revised 19 May 1995; accepted 22 May 1995. receptor. Recent work points to a role for the receptor in facilitating the dissociation of iron (Bali et al., 1991; Bali and Aisen, 1991; 1992; Egan et al., 1993). Often the specificity of the interaction of transferrin with the receptor is overlooked. Four studies in particular high- light how ineffective bovine transferrin is rela- tive to human transferrin in binding to human receptors and delivering iron to cells (Mess- mer, 1973; Penhallow et al., 1986; Tsavaler et al., 1986; Young and Garner, 1990). The effect is overcome by the large excess of bovine transferrin in the 5% to 10% fetal bovine se- rum used in standard tissue culture of human derived cells. Most serum substitutes or sup- plements contain human transferrin at a far lower concentration than is found for bovine transferrin in fetal calf serum. One result of culturing human cells in fetal calf serum for many passages is hyperexpression of trans- ferrin receptor on these cells. HelaS, cells, for example, have l-2 million transferrin recep- tors per cell (Penhallow et al., 1986). 347