Generation and characterization of an IGFBP-7 antibody: identification of 31kD IGFBP-7 in human biological fluids and Hs578T human breast cancer conditioned media.

Abstract

The cDNA encoding mac25 (IGFBP-7) was firsr derived from mRNA isolated from leptomeningial and senescent human mammary epithelial cells (1,2). The open reading frame was shown to predict a protein with homology to the amino terminus of the IGF binding proteins, (IGFBP)1-6. Studies in our laboratory have shown that baculovirus generated mac25 binds IGF-I and-II in a specific manner, leading to the renaming of mac25 as IGFBP-7 (3). Further studies at the cellular level, to identify the involvement of IGFBP-7 in IGF regulation and cell growth, require a specific antibody against the protein, which has yet to be identified in either cultured cells or in vivo. We have now generated three polyclonal antibodies against the purified baculovirus peptide and, by western immunoblots and immunoprecipitation, demonstrated the existence of a specific 31,000 dalton protein. It is a secreted protein, and can be identified in the conditioned media of Hs578T breast cancer cells, as well as in normal human urine, cerebrospinal fluid and amniotic fluid. Subsequent studies with these antibodies should help elucidate the physiological role(s) of this protein.