PURIFICATION OF ESTRAMUSTINE-BINDING PROTEIN AND PRODUCTION OF MONOCLONAL-ANTIBODIES TO ITS DIFFERENT COMPONENTS

Estramustine-binding protein (EMBP) is a heterodimeric 46-kDa glycopro tein that is secreted from the prostate. Upon reductive cleavage it de composes into two closely related components, C1 and C2, and the share d glycosylated peptide C3. EMBP binds estramustine and estromustine, t he active metabolites of estramustine phosphate (Estracy(R)), which is a drug with antimitotic properties used in the treatment of prostatic carcinoma. In the present study, a two-step procedure (i.e., anion-ex change and Con A-Sepharose chromatography) is described for the isolat ion of EMBP in high yield from rat prostate tissue. Mouse monoclonal a ntibodies (mAbs) were produced using the major DEAE-Sepharose fraction of EMBP as an immunogen. Eleven mAbs were selected by screening in a solid-phase ELISA. One displayed high-affinity binding with soluble EM BP (K-a approximate to 3 x 10(10) M(-1)) and crossreacted with a human prostate tumor extract in a radioimmunoassay. The epitopes defined by these mAbs were analyzed by Western immunoblotting. All constituents of EMBP, except component C1, were identified by at least one antibody . Nine visualized either one or both of the two EMBP subunits under de naturing conditions, two of which retained their reactivity after redu ction of disulfide bridges. One epitope was exposed to its mAb only wh en the antigen was in its reduced state. The immunoreactivity was elim inated by protease treatment, whereas deglycosylation with glycopeptid ase F had a minimal effect. EMBP has been detected in tissues other th an the prostate as well as in prostate neoplastic specimens and in sev eral other human malignancies. Hence, these mAbs will be a useful tool in the characterization of EMBP in different tissues and in evaluatin g existing and in defining new indications for Estracyt therapy. (C) 1 995 Wiley-Liss, Inc.