Single-step purification of prostatic acid phosphatase: immunoaffinity chromatography with a monoclonal antibody.
BACKGROUND
Prostatic acid phosphatase (PAP) is an important protein which should be studied further as a tumor marker or as a biologically functional molecule. The purpose of the study was to establish a simple and reliable method to obtain highly pure PAP.


METHODS
Spleen cells from mice immunized with prostatic epithelial cells prepared from benign prostatic hyperplasia tissue were fused with myeloma cells X63Ag8-653. Hybrid cells of interest were selected using the indirect immunofluorescence method with unfixed frozen tissue sections. One clone of the hybrid cell lines was established which secreted the monoclonal antibody specifically reactive to prostatic acid phosphatase. Using this monoclonal antibody, we purified the antigen from human prostatic tissue by means of single-step immunoaffinity chromatography.


RESULTS
SDS-PAGE profiling under reducing conditions indicated that the protein recognized by this antibody consisted of several components of molecular weight 41,000-45,000. Partial amino acid sequence analysis of this protein indicated that these components involved a heterogeneously modified single polypeptide, and that this antigen is identical to human prostatic acid phosphatase.


CONCLUSIONS
This single-step method saves the time needed to purify prostatic acid phosphatase and requires only half a day for the whole procedure. Moreover, the purity of the isolated protein was extremely high. This method seems to be useful not only for purifying prostatic acid phosphatase but also for purifying other proteins from the prostate gland and for analysis of antigenic macromolecules.
Yoshiki
T
T
Department of Urology, Faculty of Medicine, Kyoto University, Japan.
Ueda
M
M
Hirano
A
A
Okada
K
K
Yoshida
O
O
D000135:Acid Phosphatase / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D000918:Antibody Specificity; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011467:Prostate / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D000135:Acid Phosphatase