A solid-phase fluorescent immunoassay for human prostatic acid phosphatase.

Abstract

Abstract Prostatic acid phosphatase was extracted from human malignant prostatic tissues by Tween 80 and precipitated with 40 to 75% of ammonium sulfate. The crude extract of prostatic acid phosphatase was further purified by a series of chromatographies with concanavalin A-Sepharose, diethylaminoethyl cellulose, and Sephadex G-100. A homogeneous prostatic acid phosphatase preparation with an 85-fold purification and a recovery of 38% of initial enzyme activity was obtained. Antiserum was raised by immunizing female rabbits with the purified enzyme preparation. The anti-prostatic acid phosphatase serum did not cross-react with the acid phosphatase of other human tissues. The anti-prostatic acid phosphatase immunoglobulin G (IgG) antibody was isolated by diethylaminoethyl cellulose and conjugated to CNBr-activated Sepharose 4B, which was then used in a solid-phase fluorescent immunoassay for serum prostatic acid phosphatase. Prostatic acid phosphatase in the serum was separated from other acid phosphatases by mixing serum with antiprostatic acid phosphatase (IgG)-Sepharose 4B; the enzyme activity was subsequently measured by incubating prostatic acid phosphatase-IgG-Sepharose and α-naphthyl phosphate. The hydrolysis product, α-naphthol, is fluorogenic and can be quantitated with a spectrophotofluorometer at 340 nm (excitation) and 465 nm (emission). The sensitivity of this solid-phase immunofluorometric assay was 60 pg of prostatic acid phosphatase per ml, more sensitive than other immunoassays for prostatic acid phosphatase. Further, the antibody could be reused after the prostatic acid phosphatase was dissociated from IgG-Sepharose 4B. Normal male serum was found to contain an average of 5.619 ng of prostatic acid phosphatase per ml (from 30 normal males, range 1.399 to 9.839 ng per ml). Patients with nonprostatic cancer have normal serum prostatic acid phosphatase levels. Patients with early stages of prostate tumor had elevated levels of serum prostatic acid phosphatase.