Abstract |
Prostate specific antigen (PSA) is a commonly used marker of prostate cancer. A panel of four kallikrein immunoassays has been reported to improve the prediction of prostate biopsy outcome ( cancer vs benign) in men with elevated PSA in the circulation. Assay of one of the kallikrein forms, intact free PSA (fPSA-I), is based on a unique monoclonal antibody (4D4), which is specific for PSA without the internal cleavage at Lys(145)-Lys(146). Due to high dissociation rate the 4D4 antibody is less than optimal for achieving a highly sensitive robust assay. In this study, we cloned the 4D4 Mab into a recombinant fragment (Fab) format and constructed three mutant libraries with the aim to increase its binding affinity. The libraries contained targeted mutations either in the CDR-H1, CDR-H2 or CDR-L3 region. PSA-I specific antibodies were enriched from the libraries by phage display technology. We identified fourteen unique clones with 1-5 mutated amino acids showing reduced dissociation of the PSA conjugate compared to the wt-4D4 Fab. Five of these mutant antibodies had 2-6 times higher binding affinity compared to the wt-4D4 Fab yet retaining the original specificity for PSA-I. The analytical sensitivity of fPSA-I assay with mutant L3-2 Fab was 0.12 μg/L compared to 4.46 μg/L with the original wt-4D4 Fab. In the method comparison study, the developed assay showed an excellent correlation to the existing fPSA-I assay. The high affinity and specificity of these mutant antibodies have potential to provide sensitive and robust detection of intact and nicked PSA from patient samples in different test formats.
|
Authors | Md Ferdhos Khan Liton, Mari T Peltola, Markus Vehniäinen, Erica Kuusela, Tiina Pettersson, Urpo Lamminmäki, Kim Pettersson, Eeva-Christine Brockmann |
Journal | Journal of immunological methods
(J Immunol Methods)
Vol. 422
Pg. 72-9
(Jul 2015)
ISSN: 1872-7905 [Electronic] Netherlands |
PMID | 25889751
(Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
|
Copyright | Copyright © 2015 Elsevier B.V. All rights reserved. |
Chemical References |
- Antibodies, Monoclonal
- Immunoglobulin Fab Fragments
- Prostate-Specific Antigen
|
Topics |
- Antibodies, Monoclonal
(immunology)
- Antibody Affinity
(immunology)
- Cell Surface Display Techniques
(methods)
- Humans
- Immunoassay
- Immunoglobulin Fab Fragments
(genetics, immunology)
- Male
- Prostate-Specific Antigen
(analysis, blood, immunology)
- Prostatic Neoplasms
(diagnosis)
|