Monoclonal Antibodies to the Amino- and Carboxyl-Terminal Domains of Ovotransferrin

Abstract

Monoclonal antibodies to the iron transport protein ovotransferrin were produced by immunizing mice simultaneously with ovotransferrin and with the proteolytically derived amino- and carboxyl-terminal half-molecule domains of ovotransferrin. Two isolated hybridoma clones (designated α0T+N1 and α0T+N2) produced antibodies (IgG1) to determinants located on holo-ovotransferrin and the amino-terminal domain; two hybridoma clones (designated α0T+C1 and α0T+C2) produced antibodies (IgG₁) to determinants on holo-ovotransferrin and the carboxyl-terminal domain. One hybridoma clone (designated α0T-N1) produced an antibody (IgG₁) that bound only the amino-terminal domain and did not bind holo-ovotransferrin. Both α0T+N1, and α0T-N1 bound to antigen less tightly after removal of iron; antibodies α0T+N2, α0T+C1, and α0T+C2 were unaffected by removal of iron from holo-ovotransferrin or the isolated domains. Intact disulfide bonds in the antigens were required for binding by the antibodies. These antibodies should prove useful as probes for discrete regions of the ovotransferrin molecule, in particular, those regions involved in binding to the transferrin receptor.