Immunopeptidometric Assay for Enzymatically Active Prostate-Specific Antigen

Wu, Ping; Zhu, Lei; Stenman, Ulf‐Hǎkan; Leinonen, Jari

doi:10.1373/clinchem.2003.026146

Cited by 36 publications

(27 citation statements)

References 18 publications

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“…The selection of PSA-specific peptides has already been reported by Wu et al using a cyclic peptide library (Wu et al, 2000;Pakkala et al, 2004;Wu et al, 2004a;Wu et al, 2004b). In the present study, using both linear and cyclic libraries, we also selected PSA-specific peptides and showed that the cyclization of the peptide was not essential to obtain modulation of the PSA enzymatic activity.…”

Section: Discussionsupporting

confidence: 63%

“…We also described the characteristics of 16H9A12, another anti-PSA mAb, the epitope of which was shown to be located on the kallikrein loop (Michel et al, 2001a). However, other studies described cyclic peptides that bind specifically to non-clipped form of mature PSA and enhance its enzymatic activity (Wu et al, 2000(Wu et al, , 2004b. The authors suggested that these peptides could interact near the active site of the enzyme.…”

Section: Introductionmentioning

confidence: 98%

“…The presence of proforms of the protein and internal cleavages, for example at K145-K146 or K182-S183, are two commonly proposed explanations of free PSA in the circulation (Mikolajczyk et al, 2001). However, 1-10% of free active PSA, in serum from prostate cancer patients, has been recently detected (Niemela et al, 2002;Wu et al, 2004b).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of prostate‐specific antigen binding peptides selected by phage display technology

Ferrieu-Weisbuch

Michel

Collomb-Clerc

et al. 2005

J of Molecular Recognition

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Prostate-specific antigen (PSA) is an important marker for the diagnosis and management of prostate cancer. Free PSA has been shown to be more extensively cleaved in sera from benign prostatic hyperplasia patients than in sera from prostate cancer patients. Moreover, the presence of enzymatically activatable PSA was characterized previously in sera from patients with prostate cancer by the use of the specific anti-free PSA monoclonal antibody (mAb) 5D3D11. As an attempt to obtain ligands for the specific recognition of different PSA forms including active PSA, phage-displayed linear and cyclic peptide libraries were screened with PSA coated directly into microplate wells or presented by two different anti-total PSA mAbs. Four different phage clones were selected for their ability to recognize PSA and the inserted peptides were produced as synthetic peptides. These peptides were found to capture and to detect specifically free PSA, even in complex biological media such as sera or tumour cell culture supernatants. Alanine scanning of peptide sequences showed the involvement of aromatic and hydrophobic residues in the interaction of the peptides with PSA whereas Spotscan analysis of overlapping peptides covering the PSA sequence identified a peptide binding to the kallikrein loop at residues 82-87, suggesting that the peptides could recognize a non-clipped form of PSA. Moreover, the PSA-specific peptides enhance the enzymatic activity of PSA immobilized into microplate wells whereas the capture of PSA by the peptides inhibited totally its enzymatic activity while the peptide binding to PSA had no effect in solution. These PSA-specific peptides could be potential tools for the recognition of PSA forms more specifically associated to prostate cancer.

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Section: Discussionsupporting

confidence: 63%

Section: Introductionmentioning

confidence: 98%

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Characterization of prostate‐specific antigen binding peptides selected by phage display technology

Ferrieu-Weisbuch

Michel

Collomb-Clerc

et al. 2005

J of Molecular Recognition

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“…We purified PSA from seminal fluid and trypsin-2 from urine as described (12,13 ). We purchased API from Athens Research and Technology, Inc. and ACT from Sigma and prepared PSA-API, PSA-ACT, and trypsin-2-API as described (14 -16 ).…”

Section: Purified Proteins Antibodies and Reagentsmentioning

confidence: 99%

Proximity Ligation Measurement of the Complex between Prostate Specific Antigen and α1-Protease Inhibitor

et al. 2009

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Background: Prostate specific antigen (PSA)–α1-protease inhibitor complex (PSA-API) is a minor form of PSA in serum. It may be useful for prostate cancer (PCa) diagnosis, but its specific detection is hampered by nonspecific background. To avoid this, we developed an immunoassay for PSA-API based on proximity ligation. Methods: We used a monoclonal antibody (mAb) to total PSA (tPSA) to capture PSA, while using another anti-tPSA mAb together with an anti-API mAb as probes. We measured PSA-API by quantification of amplified DNA strands conjugated to the probes. We measured serum PSA-API in 84 controls and 55 men with PCa who had PSA concentrations of 4.0–10 μg/L. Results: The detection limit of the assay was 6.6 ng/L. The proportion of PSA-API to tPSA (%PSA-API) tended to be lower in men with PCa (2.8%) than without cancer (3.3%) but was not statistically significant (P = 0.363). When used alone, %PSA-API [area under the curve (AUC) 0.546] did not improve detection of PCa, whereas %fPSA (AUC 0.710) and the sum of %fPSA and %PSA-API (AUC 0.723) did. At 90% diagnostic sensitivity, the diagnostic specificity for cancer was not significantly better for %fPSA + %PSA-API than for %fPSA alone (36% vs 30%). Conclusions: Proximity ligation eliminated nonspecific background, enabling accurate measurement of PSA-API in serum specimens with moderately increased tPSA. The combined use of %PSA-API and %fPSA provided a modest improvement for PCa detection, but based on the current study cohort, it is uncertain whether the improvement has clinical utility. .

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“…A highly sensitive immunopeptidometric assay for enzymatically active prostate-specific antigen (PSA) was recently established by use of a capture antibody to PSA and a fusion protein of a PSA-specific peptide with glutathione S-transferase for detection. The peptide most likely recognizes the catalytic site in intact PSA as it does not recognize PSA inactivated by partial cleavage of certain peptide bonds (15 ). Thus, its specificity is similar to that displayed by single-chain dromedary antibodies.…”

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confidence: 94%