Generation of monoclonal antibodies to prostatic acid phosphatase isoenzyme 2 and application in solid-phase enzyme immunoassay.
Monoclonal antibodies specific to prostatic acid phosphatase (PAP) isoenzyme 2 were generated by using an improved hybridoma technique. After three subcutaneous immunizations and 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 by titration curve in radioimmunoassay (RIA). Three of them (PAP 1, PAP 03, and PAP 019) were shown to be over 1 X 10(9) M-1. From the results of a matrix cross-matching procedure, a pair of antibodies (PAP 03 and PAP 1) reacting with discrete antigenic determinants were identified for preparing 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 not 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 specificity for determining PAP isoenzyme 2 in human serum.
Kuo
C Y
CY
Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, Canada.
Chen
K W
KW
Fu
J
J
Lam
K W
KW
Lee
C Y
CY
D000135:Acid Phosphatase / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal; D002459:Cell Fusion; D006822:Hybrid Cells; D007114:Immunization; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis; D007527:Isoenzymes / Q000032:analysis*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D000073718:Pancreatitis-Associated Proteins; D011467:Prostate / Q000201:enzymology*; D011863:Radioimmunoassay; D012015:Reference Standards
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007527:Isoenzymes; D000073718:Pancreatitis-Associated Proteins; C000612141:REG3A protein, human; D000135:Acid Phosphatase