Purification of estramustine‐binding protein and production of monoclonal antibodies to its different components

Abstract

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.

Lipophilin, a novel heterodimeric protein of human tears

“…As the synthesis of prostatein/EMBP is also regulated by androgens [10], these features have made prostatein a valuable experimental marker. Although there is immunochemical evidence that prostatein‐like molecules exist in humans [11, 12], the sequences of these molecules have not been reported.…”

Distribution of estramustine-binding protein during postnatal development of rat brain

“…The primary mouse monoclonal antibody Mab-EMBP 1, recognizing the C3 peptide of rat EMBP (Björk et al 1995), was added to the sections for 1 h at a concentration of 0.1 mg/ml in phosphate-buffered saline (77 mM Na 2 HPO 4 , 23 mM NaH 2 PO 4 , and 0.15 M NaCl) containing 10 mg/ml bovine serum albumin. Immediately after decapitation, the brains were removed from the skull and fixed in 5-7°C 70% ethanol for more than 24 h. Before paraffin embedding, brains were divided into four parts in the coronal plane in order to allow staining of frontal, parietal, and temporal brain as well as basal ganglia, brainstem, cerebellum, and the ventricular system.…”