Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay

Tang J. Wang, H. Jay Linton, Janice Payne, Harry G. Rittenhouse, Daniel Wan-Yui Chan, Alan Wayne Partin, Robert L. Wolfert, Kristine Kuus-Reichel

Research output: Contribution to journalArticle

Abstract

The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including α1 antichymotrypsin (ACT) and α2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites. Existing immunoassays for quantitation of total serum PSA measure PSA-ACT (CPSA) and free PSA (FPSA), which comprise the major and minor components of total PSA, respectively. Monoclonal antibodies (MAb) specific for CPSA alone were generated using a unique immunogen prepared by blocking the major antigenic determinants on FPSA and ACT. The blocked immunogen greatly enhanced the frequency of hybridomas reactive against the CPSA complex. CPSA prototype immunoassays were established using anti-CPSA (PX1G359) or anti-ACT (AC1A212) MAb as tracer MAb and anti-PSA (PSA399) MAb as capture MAb. The complex-selective MAbs demonstrated minimal cross- reactivity with Cathepsin-G (CG) ACT (CG-ACT), ACT or FPSA. CG-ACT complex interfered with the accuracy of initial prototype assays specific for CPSA measurement and caused over-recovery (1 to 3 ng/mL, with 40 to 180 ng/mL range of CG-ACT in serum) of (apparent CPSA values. Addition of 0.4% NP-40 and 0.1% 0.088 micron microparticles in clinical specimens eliminated this interference. Specimens from 39 prostate cancer (PCa) patients and 44 benign prostatic hyperplasia (BPH) patients were analyzed with the PX1G359 CPSA assay. In this study, the area under the curve (AUC) values for ROC analysis of total PSA (CPSA+FPSA), FPSA to total PSA ratio (f/t), and FPSA to CPSA ratio (f/c) were 0.357, 0.634, and 0.624, respectively. In a second study using AC1A212 CPSA assay, where specimens from 16 PCa patients and 48 BPH patients were tested, the AUC values for total PSA, f/t and f/c ratios were 0.62, 0.785, and 0.732, respectively. Using the CPSA assay with minimal interference our studies are consistent with previous CPSA data showing that the f/t PSA ratio remains superior to the f/c PSA ratio in differentiating PCa and BPH diseases. Complex PSA by itself or as ratio with free or total PSA does not provide any advantage over the established method of FPSA to total PSA ratio.

Original languageEnglish (US)
Pages (from-to)535-541
Number of pages7
JournalHybridoma
Volume18
Issue number6
StatePublished - Dec 1999

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Prostate-Specific Antigen
Immunoassay
Monoclonal Antibodies
Cathepsin G
Macroglobulins
Prostatic Neoplasms
Prostatic Hyperplasia
Area Under Curve
Serum
Prostatic Diseases
Antibody Binding Sites
Serine Proteinase Inhibitors
Hybridomas
Masks
ROC Curve

ASJC Scopus subject areas

  • Immunology
  • Genetics

Cite this

Wang, T. J., Linton, H. J., Payne, J., Rittenhouse, H. G., Chan, D. W-Y., Partin, A. W., ... Kuus-Reichel, K. (1999). Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay. Hybridoma, 18(6), 535-541.

Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay. / Wang, Tang J.; Linton, H. Jay; Payne, Janice; Rittenhouse, Harry G.; Chan, Daniel Wan-Yui; Partin, Alan Wayne; Wolfert, Robert L.; Kuus-Reichel, Kristine.

In: Hybridoma, Vol. 18, No. 6, 12.1999, p. 535-541.

Research output: Contribution to journalArticle

Wang, TJ, Linton, HJ, Payne, J, Rittenhouse, HG, Chan, DW-Y, Partin, AW, Wolfert, RL & Kuus-Reichel, K 1999, 'Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay', Hybridoma, vol. 18, no. 6, pp. 535-541.
Wang, Tang J. ; Linton, H. Jay ; Payne, Janice ; Rittenhouse, Harry G. ; Chan, Daniel Wan-Yui ; Partin, Alan Wayne ; Wolfert, Robert L. ; Kuus-Reichel, Kristine. / Generation of PSA-ACT-specific monoclonal antibodies and their application in a sandwich immunoassay. In: Hybridoma. 1999 ; Vol. 18, No. 6. pp. 535-541.
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abstract = "The prostate-specific antigen (PSA) immunoassay is an important tool for the detection and monitoring of prostate cancer. PSA exists in serum mainly as complexes with serine protease inhibitors including α1 antichymotrypsin (ACT) and α2 macroglobulin (MG). PSA-MG complex is not detected by the existing PSA immunoassays since MG (720 kDa) sequesters PSA and masks the antibody binding sites. Existing immunoassays for quantitation of total serum PSA measure PSA-ACT (CPSA) and free PSA (FPSA), which comprise the major and minor components of total PSA, respectively. Monoclonal antibodies (MAb) specific for CPSA alone were generated using a unique immunogen prepared by blocking the major antigenic determinants on FPSA and ACT. The blocked immunogen greatly enhanced the frequency of hybridomas reactive against the CPSA complex. CPSA prototype immunoassays were established using anti-CPSA (PX1G359) or anti-ACT (AC1A212) MAb as tracer MAb and anti-PSA (PSA399) MAb as capture MAb. The complex-selective MAbs demonstrated minimal cross- reactivity with Cathepsin-G (CG) ACT (CG-ACT), ACT or FPSA. CG-ACT complex interfered with the accuracy of initial prototype assays specific for CPSA measurement and caused over-recovery (1 to 3 ng/mL, with 40 to 180 ng/mL range of CG-ACT in serum) of (apparent CPSA values. Addition of 0.4{\%} NP-40 and 0.1{\%} 0.088 micron microparticles in clinical specimens eliminated this interference. Specimens from 39 prostate cancer (PCa) patients and 44 benign prostatic hyperplasia (BPH) patients were analyzed with the PX1G359 CPSA assay. In this study, the area under the curve (AUC) values for ROC analysis of total PSA (CPSA+FPSA), FPSA to total PSA ratio (f/t), and FPSA to CPSA ratio (f/c) were 0.357, 0.634, and 0.624, respectively. In a second study using AC1A212 CPSA assay, where specimens from 16 PCa patients and 48 BPH patients were tested, the AUC values for total PSA, f/t and f/c ratios were 0.62, 0.785, and 0.732, respectively. Using the CPSA assay with minimal interference our studies are consistent with previous CPSA data showing that the f/t PSA ratio remains superior to the f/c PSA ratio in differentiating PCa and BPH diseases. Complex PSA by itself or as ratio with free or total PSA does not provide any advantage over the established method of FPSA to total PSA ratio.",
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