Purification of estramustine-binding protein and production of monoclonal antibodies to its different components.
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.
Björk
P
P
Department of Immunology, Pharmacia Oncology Immunology, Sweden.
Donn
F
F
Glad
C
C
Sundblad
G
G
Vestberg
M
M
Kalland
T
T
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D003429:Cross Reactions; D004961:Estramustine; D005260:Female; D016056:Immunodominant Epitopes; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011467:Prostate; D029584:Prostatic Secretory Proteins; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D016056:Immunodominant Epitopes; D029584:Prostatic Secretory Proteins; C028321:estramustine-binding protein; D004961:Estramustine