Generation of Monoclonal Antibodies to Prostatic Acid Phosphatase Isoenzyme 2 And Application in Solid‐Phase Enzyme Immunoassay

Abstract

Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three intravenous boosters, cell fusion experiments were performed. The hybrid cells were first cultured in a semisolid medium containing methylcellulose and later transferred to a liquid medium for further subculture. Out of a total of 600 colonies recovered after two cell fusion experiments, 13 were shown to exhibit affinity to PAP isoenzyme 2 by radioimmunoassay. Nine hybrid cell lines which showed high affinity and specificity were established for further evaluation. Their immunoglobulin subclass was determined to be immunoglobulin G. The association constants between PAP isoenzyme 2 and each monoclonal antibody were determined titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 .times. 109 M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic deteminants were identified for perparing a solid phase sandwich enzyme immunoassay (EIA) kit. The designed EIA procedure could be performed within 40 min in a one-stage incubation protocol. The assay time was shorter than that of other commercial RIA or EIA kits, and the sensitivity was 0.4 ng/ml which was comparable to that of RIA kits. The EIA kit was shown to cross-react with human thyroid stimulating hormone, .alpha.-fetoprotein, carcinoembryonic antigen, and acid phosphatases derived from tissues other than prostate. Therefore, this design was a simple and rapid method with high sensitivity and specifically for determining PAP isoenzyme 2 in human serum.