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Solid‐phase immunofluorescent and immunoadsorbent assays of serum prostatic acid phosphatase
Author(s) -
Lee C. L.,
Wang M. C.,
Killian C. S.,
Slack N. H.,
Murphy G. P.,
Chu T. M.
Publication year - 1980
Publication title -
the prostate
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.295
H-Index - 123
eISSN - 1097-0045
pISSN - 0270-4137
DOI - 10.1002/pros.2990010406
Subject(s) - prostatic acid phosphatase , acid phosphatase , antiserum , chemistry , phosphatase , microbiology and biotechnology , sepharose , enzyme , immunoassay , chromatography , prostate , biochemistry , antibody , cancer , medicine , biology , immunology
Human prostatic acid phosphatase was purified to homogeneity from malignant prostatic tissue by Tween 80 extraction and 40‐75% ammonium sulfate precipitation, followed by Con A‐Sepharose, DEAE‐cellulose, and gel filtration chromatography. A specific antiserum was raised by immunizing female goats or rabbits with the purified enzyme. This antiserum did not cross‐react with the acid phosphatase of other human tissues. Two immunochemical methods, a solid‐phase fluorescent immunoassay and a solid‐phase immunoadsorbent assay, were developed. The IgG antibody fraction from antiprostatic acid phosphatase was conjugated to CNBr‐activated Sepharose 4B, which was then used in the two immunoassays to separate serum prostatic acid phosphatase from other acid phosphatases or serum proteins. The enzyme activity was subsequently measured by incubating the solid‐phase bound prostatic acid phosphatase with α‐naphthyl phosphate and quantitating the fluorescent product with a spectrophotofluorometer (immunofluorometric assay) or quantitating the α‐naphthol‐FRBS colored complex with a spectrophotometer (immunoadsorbent assay). The sensitivity of this immunofluorometric assay was 60 pg/ml, more sensitive than other immunoassays. The results obtained from clinical evaluation indicate that serum prostatic acid phosphatase in prostate cancer can be detected in significant percentage with early stages of prostatic cancer. The sensitivity of the immunoadsorbent assay was 0.22 IU/l of enzyme activity or 0.88 ng of prostatic acid phosphatase protein per ml serum. Initial clinical evaluation demonstrated that 19 of 25 patients with early stages of prostate cancer and 12 of 14 patients with metastatic prostate cancer exhibited an elevated serum PAP level (over all 79%), as compared with only six and eight patients respectively (overall 35%), by a conventional chemical method.