Estramustine-binding protein (
EMBP) is a heterodimeric 46-kDa
glycoprotein that is secreted from the prostate. Upon reductive cleavage it decomposes into two closely related components, C1 and C2, and the shared glycosylated
peptide C3.
EMBP binds
estramustine and
estromustine, the active metabolites of
estramustine phosphate (
Estracyt), 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-exchange and
Con A-Sepharose chromatography) is described for the isolation of
EMBP in high yield from rat prostate tissue. Mouse
monoclonal antibodies (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
EMBP (Ka approximately 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 denaturing conditions, two of which retained their reactivity after reduction of
disulfide bridges. One
epitope was exposed to its mAb only when the
antigen was in its reduced state. The immunoreactivity was eliminated by
protease treatment, whereas deglycosylation with
glycopeptidase F had a minimal effect.
EMBP has been detected in tissues other than the prostate as well as in prostate neoplastic specimens and in several other human
malignancies. Hence, these mAbs will be a useful tool in the characterization of
EMBP in different tissues and in evaluating existing and in defining new indications for
Estracyt therapy.